Holzhauer won $80,615 on Thursday's episode, bringing his total to $1,608,627.
The native of Naperville on Wednesday tied for the title with Julia Collins, who in 2014 collected $428,100 over 20 games on the quiz show, before claiming it for his own one day later.
"'What is 'The Glass Menagerie?" was the correct answer. His total sits now at $1,528,012, which is more than halfway to Jennings' total of $2.5 million.
Ken Jennings is now the only contestant left for Holzhauer to beat.
He won $101,682 on Wednesday night.
"It is exciting for me to see the stats guys winning the battle and getting into Major League Baseball front offices", Holzhauer said. He now has a ceremonial key to the Las Vegas Strip. His unique playing style - seeking out high-value clues first and going "all-in" on daily doubles - has made him a crowd favorite.
Holzhauer planned his strategy from the beginning, only deviating from it if the category is one he's unfamiliar with - such as the U.S. Senate.
He first broke the single-game winnings record in April, when he earned $110,914.
'This is absolutely insane, ' Jennings said on Twitter of Holzhauer's success. "I don't know if I'm the kind of networking/interpersonal skills guy who could become a GM, but being a guy who can help the team on making decisions on which guys to trade for and sign as free agents, that was the dream back then".