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In the hillbilly community of Dogpatch, the local women eagerly await the upcoming running of the annual Sadie Hawkins Day race, during which they can catch a husband.  The most determined woman is Daisy Mae, who has chased after the handsome, carefree Li’l Abner Yokum every year without success.  Abner’s parents, Pappy and Pansy “Mammy” Yokum, are hopeful that Daisy Mae will catch Abner, whom Mammy daily doses with her own “Yokumberry tonic” to make him strong and healthy.  Unfortunately for Daisy Mae, Earthquake McGoon, who has recently been crowned “world champion dirtiest wrestler,” desires to marry her himself, even though, according to the “code of the hills,” they cannot be wed unless she catches him in the race.

Hoping to find a solution to his dilemma, Earthquake allies himself with the incompetent Senator Jack S. Phogbound, who, at a village meeting, reveals to everyone that he has discovered a way for Dogpatch to become famous.  Phogbound explains that in order to protect Las Vegas from fallout from nearby atomic bomb testing, the U.S. government decided to find the “most unnecessary” town in the country instead to “blow off the face of the earth” with an atom bomb.  Dogpatch, having been determined to be the most useless place in the U.S., has been chosen, and so the citizens must evacuate immediately, according to visiting scientist Dr. Rasmussen T. Finsdale.

At first all are pleased by their new distinction until they realize that they will have to leave Dogpatch before the race is held.  Daisy Mae is especially horrified when Mayor Daniel D. Dawgmeat confirms that without the race, any male who obtains the consent of a female’s kinfolk may claim her in marriage.  Daisy Mae’s worthless relatives, the foul-smelling and evil-tempered Scragg clan, have already accepted a bribe from Earthquake, and so she will be forced to marry him.  The infuriated Mammy and Pappy lead their friends in attempting to find something that will prove Dogpatch is necessary, but even when storekeeper Available Jones shows Finsdale the statuesque Stupefyin’ Jones, whose every wiggle can stupefy a man into near-unconsciousness, the scientist dismisses their efforts.  Daisy Mae is encouraged, however, as Abner had proposed to her when Earthquake tried to establish his matrimonial claim.

When the local Indians give Finsdale’s assistant a sample of their Kickapoo Joy Juice, the powerful beverage scorches his throat, so he gratefully takes a swig of Mammy’s Yokumberry tonic.  Everyone is astonished when the skinny assistant is transformed into a muscle-bound youth, just like Abner, prompting Finsdale and the military representatives to decide to take the tonic, Abner and several other Dogpatch men to Washington, DC for testing.  If the tonic is a success, Dogpatch will be saved, as the only tree bearing Yokumberries grows in Mammy’s yard.

The tonic appears to be a sensation, and when Abner announces his intention to give it to the U.S. government for free, private businessmen are irritated.  Especially irate is the rapacious General Bullmoose, who believes that he can make a fortune with the Yokum drug.  Bullmoose orders his “executive secretary,” the dimwitted Appassionata Von Climax, to romance Abner, but even though Abner is awed by Appassionata’s charms, he insists on giving away the secret formula.  When Abner reveals that he is going home for the next day’s race, Bullmoose decides to enter Appassionata in the race and arrange for her to catch and marry Abner, which will then entitle her to half his property, or more if she “accidentally” becomes a widow.

Back in Dogpatch, Abner tells Daisy Mae that although he hopes that she will catch him, he will not be able to help but run as quickly as possible.  Dismayed, Daisy Mae arranges with Available for the services of Stupefyin’ during the race in the hopes that she will stop Abner long enough for Daisy Mae to catch him.  When Bullmoose arrives, however, Jones sells him the use of Evil Eye Fleagle, a twitchy villain whose “whammy” renders the recipient incapable of movement.  Bullmoose intends to have Fleagle whammy Abner so that Appassionata can catch him, and during the race, despite the best efforts of Daisy Mae and Stupefyin’, Fleagle and Appassionata are triumphant.  Masking her heartbreak, Daisy Mae acknowledges that Abner now belongs to her rival, and Abner is taken back to Washington, DC.

Soon after, a worried Mammy conjures up a vision revealing Bullmoose’s plans to have Fleagle “whammy” Abner into committing suicide after inducing him to reveal the secret tonic formula to Appassionata.  Frantic, Daisy Mae agrees to marry Earthquake if he rescues Abner, and so the couple, along with preacher Marryin’ Sam, the Yokums and other Dogpatchers, race to DC, where they crash a fancy party that Bullmoose is throwing for the engaged couple.  Although Abner refuses to listen to Daisy Mae’s assertions about Appassionata’s intentions, Earthquake saves the day by using a tray to deflect Fleagle’s truth-telling whammy onto Bullmoose.

When Mammy then questions the General, he confirms his nefarious plans.  Bullmoose and Appassionata are arrested, but just when the hillbillies think that they are safe, military officers announce that the evacuation of Dogpatch will go through as planned because the tonic is a failure.  When Mammy and the others rush to the laboratory for an explanation, Finsdale demonstrates that the tonic, while making men physically perfect and eternally youthful, robs them of any romantic feelings.  Mammy is especially distraught, as she realizes that it is her fault that Abner cannot love Daisy Mae.  Finsdale insists that Abner stay for further study, but after the others leave, Pappy assures him that there is another potion that can correct his problem, on the condition, that he truly wants it.  Abner assures him that he does, and so Pappy wires Sam to stall Daisy Mae and Earthquake’s wedding.

The next day, Sam does his best to prolong the ceremony, and Daisy Mae helps by bringing in all of her various Scragg relatives, claiming that they want to live with her and her rich husband.  Disgusted by the Scraggs, Earthquake readily cedes Daisy Mae’s hand to Abner when he arrives suddenly.  Pappy gives Abner a swig of his special potion, which, he confides to Mammy, is merely creek water.  Because of Abner’s belief, however, the liquid works and he happily agrees to wed Daisy Mae.  Before the ceremony can be completed, however, the military demands that the evacuation begin.  When the hillbillies then attempt to dismantle the statue of Jubilation T. Cornpone, Dogpatch’s founder, they uncover a tablet written by Abraham Lincoln, declaring that because of Cornpone’s ineptitude in the Confederate Army, the North was able to win the Civil War.  With the statue pronounced a national monument, the town is saved from being blown up, and Daisy Mae and Abner celebrate with an enthusiastic kiss.