The stage was set for the opening showdown of the trials in the morning heats when Kalisz narrowly pipped Lochte at the wall but the 22-year-old left no doubt in the final as he pulled away from the 2012 London gold medallist over the final two legs clocking four minutes, 9.54 seconds.
"I'm glad to get this one out of the way, this one for me was a lot more stressful than I could imagine Rio could be," Kalisz, who is headed to his first Olympics, said after becoming the second fastest in the world this year.
"This is the one thing I wanted to accomplish, anything after that is going to be a bonus.
"I've got a lot more in the tank going forward to Rio."
Lochte led by nearly two lengths after the butterfly and backstroke.
But he faded badly in the back half to finish third as 20-year-old Jay Litherland powered past the five-time Olympic champion in the freestyle final leg to also likely grab a Rio berth.
The winner in each event automatically qualifies for Rio in August, with second place finishers also usually earning tickets.
Lochte, an 11-time Olympic medallist seeking to make his fourth U.S. team, said after the race he had pulled his groin in the morning heat, leaving some question as to whether he will be able to compete in all his races.
"I thought about it this morning scratching but you know, it's the Olympic trials," said Lochte. "If I had broken a leg I would still go out there and swim.
"I'm going to keep working on it day in and day out and hopefully it gets better."
Michael Phelps, who dropped the 400 IM in which he still owns the world record, clearly felt no remorse about giving up what is considered swimming's toughest test, smiling as he arrived on the pool deck to watch his old foe Lochte and training partner Kalisz battle.
"I was so happy for Chase, I was crying when I was hugging him," said Phelps, who now trains in Arizona with Kalisz under coach Bob Bowman.
"He is like my brother and watching him to be able to do that is a very special moment.
"I know Chase is very determined. I've trained with him every day and the kid works his butt off."
Eighteen-time Olympic champion Phelps begins his bid for a fifth and what he says will be his final Olympic team on Tuesday with the 200 freestyle.
Opening night featured two other finals at the soldout CenturyLink Center with Maya DiRado qualifying for her first Olympic team with a victory in the women's 400 IM holding off 2012 silver medallist Elizabeth Beisel.
Connor Jaeger, who narrowly missed out on a medal in the 400 freestyle at last year's world championships finishing fourth, will have a shot at the Olympic podium after taking the evening final ahead of Conor Dwyer.
(Editing by Andrew Both)
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