Jefferson made a soft 15-foot jump shot with 16.9 seconds left for a 65-64 Hogs lead, then blocked Jeff Hawkins' corner shot on the other end with 2.5 ticks to go.
The Jayhawks (1-2) had one more chance, but Arkansas center Steven Hill blocked Russell Robinson's corner attempt and Eric Ferguson secured the ball as the buzzer sounded on the Razorbacks' 65-64 win -- their biggest of the Stan Heath coaching era.
Ronnie Brewer scored 17 of his 23 points in the second half for Arkansas (2-1) and made the pass that set up Jefferson's game-winner -- the 20th lead change of an NCAA Tournament-quality matchup.
"We were looking for Ronnie or Jonathon Modica on the last shot, but teams know who's who and Kansas gave Ronnie a lot of attention," Heath said. "He made the play that set up Dontell for a nice 15- to 17-footer and Dontell knocked it down."
Jefferson had 13 points, 5 assists, 4 steals and 2 blocked shots, including the biggest of his career.
"I just helped out inside and then contested the shot on the kick-out," Jefferson said, modestly. "I'm just happy we won the game."
Kansas had called its last timeout with 12.5 seconds to go, after which Heath piggybacked and also used his final timeout.
"We debated showing Kansas something different from man-to-man," Heath said. "We knew they're young. But the consensus was, 'Hey, let's get it done with man.' It's tough to make adjustments with limited film, but we switched matchups and tried to take away the things they might do."
Kansas coach Bill Self, who had earlier set up two alley-oop dunks in timeouts, said, "On the last possession we did a very poor job. Give Arkansas credit -- they made the play. We wanted to clear the side for Brandon Rush (team-high 20 points) for a pick-and-pop, but we came off unaggressive.
"We threw the ball to our point guard in the corner. We executed well until the last part of the sequence, but it's my fault for not telling them to drive and score."
Self credited Rush, a freshman, with a good job of helping limit Brewer to 7-of-21 shooting. But he said the Jayhawks gave Brewer four to six points on free throws by fouling him away from the ball.
Down the stretch, Brewer made 4-of-4 free throws on two one-and-one chances, each time giving Arkansas the lead.
"If you're going to make the NCAA Tournament, you've got to beat good teams like Kansas and Connecticut," Brewer said. "We lost to Connecticut, but I don't think our team last year would have beaten Kansas."
Heath said the Hogs felt the lingering effects of their 77-68 loss to UConn on Monday night, but were able to overcome their fatigue.
"This was a big gut-check game for us," Heath said. "I thought Kansas wanted the game more in the first half, but in the second half I thought we wanted it more."
The proof came on the boards, where Kansas had a 20-14 rebounding edge at halftime but an 18-13 disadvantage in the second half.
Darian Townes led Arkansas with seven rebounds off the bench, while Modica quietly gathered six rebounds to go with his eight points, three assists and zero turnovers.
Heath started Hill ahead of Townes because he had not been pleased with Arkansas' defensive post play against Connecticut.
"We gave up post position in that game," Heath said. "Today Steven played smart, showed his hands and met his man early."
Freshman guard Mario Chalmers scored 11 points for Kansas and had just one of his team's 16 turnovers.
Brewer credited Arkansas senior guard Eric Ferguson with helping the team's intensity this week, even though Ferguson had five turnovers Tuesday including two down the stretch when Arkansas failed to hold a 57-49 lead.
Townes scored 10 points and played alongside Hill for several minutes against KU's tall timber up front.
"When you see the size of these teams, you almost have no choice," Heath said.
Heath was so happy that he trembled a bit during the postgame TV chat and then shook hands with several Michigan State players as he headed off the court.
Now Arkansas has to wake up early to play Maryland at 9 a.m. Hawaii time today (1 p.m. CST).
The Terrapins (2-1), jolted by Gonzaga on Monday, fell 11 points down to Chaminade on Tuesday and still trailed 46-44 at halftime before wearing down the Silverswords 98-69.
"Maryland executed their flex offense very well in the second half," Heath said. "Gary Williams' teams play very hard, so we'll need to get our rest."
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