Cousins Bo (Seann William Scott), Luke (Johnny Knoxville), and Daisy Duke (Jessica Simpson) run a moonshine business for their Uncle Jesse (Willie Nelson) in the fictional Hazzard
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Cousins Bo (Seann William Scott), Luke (Johnny Knoxville), and Daisy Duke (Jessica Simpson) run a moonshine business for their Uncle Jesse (Willie Nelson) in the fictional Hazzard County, Georgia. The cousins' primary mode of transportation is an orange 1969 Dodge Charger that the boys affectionately refer to as 'The General Lee'. Along the way, the family is tormented by corrupt Hazzard County Commissioner Jefferson Davis Hogg, widely known as "Boss Hogg" (Burt Reynolds), and his willing but dimwitted henchman, Sheriff Rosco P. Coltrane (M.C. Gainey).
After Rosco has the General Lee wrecked during Bo's attempt at winning his fifth consecutive local road rally, Billy Prickett (James Roday), a famous stock-car driver, enters Hazzard to participate in the rally. Meanwhile, Rosco plants a fake moonshine still ("'cause he's too dumb to find the real one") in Uncle Jesse's barn and seizes the Duke property in the interest of eminent domain for Boss Hogg, forcing the family to temporarily reside with neighbor and Uncle Jesse's love-interest, Pauline (Lynda Carter). Pauline informs the Dukes that Rosco seized another farm on charges so Bo and Luke investigate a local construction site and find geologic core samples with the help of bait-shop owner Sheev (Kevin Heffernan). Meanwhile, Coltrane makes arrangements to seize the General Lee as "evidence" from the local auto body shop run by the Dukes' friend Cooter (David Koechner), who instead turns the car into a hot-rod and applies a new paint job and horn, in return for finally getting payment for all the work he has done ("...'cause that's how this works...") for the boys in the past.
After retrieving the General Lee before Rosco can, the Dukes go to Atlanta to visit a local university geology lab, meeting up with Katie-Lynn Johnson (Nikki Griffin), a Hazzard county girl and the Dukes' love interest, and her Australian roommate Annette (Jacqui Maxwell). At the lab, they discover Boss Hogg's intentions of turning the county into a strip coalmine. They are later arrested by Atlanta Police after running from campus police. Back in Hazzard, Daisy learns, with the help of Sheriff Deputy Enos Strate (Michael Weston), that Billy Prickett has been hired by Boss Hogg to participate in the Rally as a ringer. Boss Hogg then heads to Atlanta where he informs the Duke boys, in lock-up, that they are too late to stop him and reveals that the vote on Hogg's proposition is at the same time as the rally, explaining Billy Prickett's involvement. During a transfer from detainment, Daisy helps the boys escape from the patrol car and they speed home to try to inform the townsfolk, escaping Atlanta Police, and the Georgia State Patrol after Bo out-maneuvers the city cops.
Upon returning home, the Dukes discover that Rosco has taken Uncle Jesse and Pauline hostage, an obvious trap for the boys, and that Billy is in on the scheme because he's ashamed of the town's low status. The two race to the farmhouse to cause a distraction to the waiting Hazzard County Sheriff's Deputies and Georgia State Troopers while Daisy and Cooter rescue Jesse and Pauline. Meanwhile, the college girls head to the rally with Sheev to inform the townsfolk about the vote on the strip-mining ordinance. Because of Sheev's armadillo hat and lack of pants, no one listens, so Bo leaves for the rally while Luke and Jesse team up to foil the county and state police who are chasing Bo, interfering with the race. Upon crossing the finish line first, before Billy, the two continue racing back and forth all the way into town, leading the townsfolk to the courthouse just in time to vote against Boss Hogg's proposed ordinance. At the courthouse, Daisy takes advantage of the governor of Georgia's presence and TV cameras to convince him into pardoning the boys, so Uncle Jesse takes the opportunity to knock out Boss Hogg and gets a pardon for assaulting a county commissioner at the same time.
The final scene shows a cook-out at the Dukes' house where Pauline convinces Uncle Jesse, who could not be found because he was "using the meat smoker," to get up and play the television series' main theme. Bo and Luke are romantically involved with the girls in the General Lee when they are caught by Luke's other love-interest Laurie Pullman (Alice Greczyn) from the intro of the film, who proceeds to chase them with a shotgun as they drive away.