Chambers clocked 10.06 seconds, his fastest time this season, to win his 100m heat at the Alexander Stadium in Birmingham and qualify for the semi-final as by far the quickest qualifier.
Meanwhile, across the continent in Finland, Gemili, beaten by Chambers at last year's British Championships, added the European U23 100m title to the world junior one he won last year.
However Gemili does not have the A standard for the World Championships unlike Chambers, who ducked under with his 10.06, and James Dasaolu who was joint-fourth fastest in Birmingham, with selections to be confirmed on Monday.
Joel Fearon, another with the A standard, was second quickest in 10.27, Mark Lewis-Francis third in 10.29 before Dasaolu and Harry Aikines-Aryeetey and Chambers is adamant he can remain unbeaten.
"That actually came as a surprise. I don't want to take anything away from these individuals, it was good for them to get out here," said Chambers.
"In athletics anything can happen. I was pleased to be able to come out here and turn things around.
"I've got the qualifying time out of the way and that's the main thing. I've still got the likes of Harry and James to come.
"I have to have more in the tank. At the end of the day, you've got to be able to push on in the semi and the final.
"I said I want to try to do my best to secure my place on the World Championship team. I'm confident of defending my title, but at the same time I want to be comfortable with what I'm doing.
"It's one thing defending a title and running OK here, but we still have to stand up to the best guys in the world and see what they're doing."
Meanwhile Dai Greene and Rhys Williams are set for a tantalising head-to-head in the 400m hurdles after qualifying for the final as the two fastest from the heats.
Williams was the quickest in 50.37, Greene 0.14 behind, while Andrew Lemoncello produced a stunning sprint finish to win the first title of the 2013 British Championships in the men's 10,000m.
Alyson Dixon was placed in the men's race after a serious of withdrawals in the women's 10,000m and, running solo with the British title assured, she clocked a time of 34:46.75 minutes.
In-form Jessica Judd was the quickest into the 800m final but not by much ahead of Marilyn Okoro, just three hundredths of a second separated the two as they were drawn apart.
Olympic gold and silver medallist Christine Ohuruogu was the fastest qualifier for the women's 400m final by almost a second and where she will be joined by younger sister Victoria.
Elsewhere British indoor 60m champion Asha Philip was the fastest qualifier in the women's 100m heats, Michael Bingham in the men's 400m and David Bishop in the men's 1500m.