Kenny Bednarek got off to a speedy start and looked as though he would pull off an upset with 50 metres to go but double world champion Lyles picked up the pace in a thrilling sprint to the finish.
Lyles crossed the line 0.06 seconds ahead of Tokyo silver medallist Bednarek, while Erriyon Knighton took third in 19.77.
Lyles, who won the men's 100m on Sunday, promised the trio would give international challengers a run for their money at the Paris Games.
"USA is the USA," Lyles said in televised remarks. "We don't give anything - you've got to take from us."
Thomas put on a clinical performance on the women's side in 21.81 seconds, the second-fastest time of the year, while Sha'Carri Richardson's hopes for an Olympic double were dashed when she finished fourth.
Tokyo bronze medallist Thomas left no doubt that the U.S. title was hers as she grabbed the lead off the turn, leaving Brittany Brown and McKenzie Long to battle with Richardson for the last two spots on the podium.
Richardson, who won the 100m a week earlier, could not match their intensity and ran out of steam down the home stretch, crossing the finish in 22.16 as Brown (21.90) and Long (21.91) each booked a spot in their first Games.
"I'm just ecstatic," said Thomas, who set a new world lead in Friday's semi-final. "Just such an amazing race."
Olympic champion Sydney McLaughlin-Levrone advanced to the 400m hurdles final with new a world lead of 52.48, jogging across the finish 1.68 seconds ahead of her rival, Rio gold medallist Dalilah Muhammad.
Alaysha Johnson had the fastest time of the 100m hurdles semi-final heats in 12.36, as Tokyo silver medallist Keni Harrison (12.52) and 2019 world champion Nia Ali (12.55) also advanced.
Lolo Jones, who returned to the trials for the first time in 12 years, finished last in her heat in 14.50.
Tara Davis-Woodhall, the favourite in the long jump, gave her fans a scare as she fouled on her first two attempts in the final and was visibly shaken as she recorded a third attempt of 6.64 metres, far below her best.
The world indoor champion regained her composure for a winning jump of 7.00m on her fifth try, however, as Jasmine Moore (6.98) finished second and Monae' Nichols (6.86) took third.
World champion Chase Jackson produced her best work of the season to win the women's shot put at 20.10 metres while Olympic silver medallist Raven Saunders finished second with 19.90 on her final try and Jaida Ross was third with 19.60.
Andrew Evans won the men's discus with a best throw of 66.61 metres on his third attempt as Sam Mattis (66.07) and Joseph Brown (65.79) rounded out the podium.
Weini Kelati won the women's 10,000 metres in 31:41.07, while Parker Valby was second (31:41.56) and Karissa Schweizer third.
The U.S. Olympic track and field trials conclude on Sunday.