Needing a field goal to extend the game or a touchdown to win, Chiefs' quarterback Patrick Mahomes orchestrated the match-winning drive to end the game with a touchdown pass to Mecole Hardman Jr.
The 49ers were leading by three in overtime after they opted for a field goal to go ahead 22-19, but were unable to get the decisive stop.
It was the second time in NFL history a Super Bowl went to overtime and the first post-season game decided under the league's new overtime rules.
Mahomes finished with 333 throwing yards, two touchdowns and 66 running yards, while 49ers quarterback Brock Purdy threw for 255 yards with one touchdown.
Mahomes, 28, was awarded the MVP Award for the third time in his career and becomes the fourth starting quarterback to win three career Super Bowls.
The only points in the first quarter came through kicker Jake Moody, who made Super Bowl history with the longest made field goal at 55 yards.
Disaster struck 49ers' linebacker Dre Greenlaw early in the second quarter when he went down with a suspected Achilles injury while running onto the field and was immediately ruled out of the game.
But not long after, a special trick-play led to the first touchdown of the game, with Purdy passing across field to wide-receiver Jauan Jennings before he threw a pass to Christian McCaffrey who ran through to the endzone untouched.
The 49ers restricted Travis Kelce to just one touch for one yard in the first half and sacked Mahomes twice, with the 49ers ahead 10-3 at halftime.
Alicia Keys, Lil John and Ludacris joined Usher during the half-time performance, while Taylor Swift was among celebrities including Blake Lively, Lady Gaga, Beyonce and Jay Z watching from the stands.
As play resumed, Chiefs kicker Harrison Butker broke Moody's Super Bowl record with a 57-yard field goal before a fumble error gave the Chiefs a huge chance for their first touchdown.
Mahomes found wide-receiver Marquez Valdes-Scantling with a 16-yard touchdown pass to take the lead for the first time in the game late in the third quarter.
However the 49ers hit back – with Purdy finding Jennings this time to score his own touchdown – however the bonus point was blocked, giving the 49ers a three-point lead with 11 minutes left.
A field goal to Butker tied the game once again, before Moody struck back to give the 49ers the lead again with less than two minutes left.
However, Butker finished regulation time with a field goal with six seconds left to tie the game 19-19.
The last team to win back-to-back Super Bowls Tom Brady's New England Patriots in 2004 and 2005.
