De Grasse had cocked his head over towards Gatlin three times before turning on the afterburners to edge the American in the heats.
The battle between Gatlin, the 35-year-old 2004 Olympic 100m champion who has served two doping bans, and Usain Bolt's self-professed successor Andre de Grasse did not play out, the Canadian deprived of the chance to run his leg after a botched handover by his teammates.
De Grasse, a three-time medallist at the Rio Olympics, had cocked his head over towards Gatlin three times before turning on the afterburners to edge the American in the heats.
But there was to be no showboating come the final at a breezy, half-full Thomas A. Robinson stadium as Aaron Brown fluffed his handover to Brendon Rodney.
The US were not out of danger, however, with Britain looking to be ahead as Gatlin took the baton from Ronnie Baker after slick passovers between Leshon Collins and Mike Rodgers.
But Englishman Danny Talbot failed to hit his mark and Gatlin had a free run in for 38.43sec, eyes glued on the big screen in near bewilderment as Barbados took silver at a distant 39.12 and China bronze (39.22).
"I'm proud of my guys, the team," said Gatlin, dubbing himself the "official track vampire" for his longevity.
"They were hungry, not nervous or jittery."
Gatlin added that the result was perfect ahead of the August world championships in London.
"We wanted to prepare ourselves, saying let's have smooth handoffs, be as fast as we can for April," he said.
"We want to come across the line first in August as we did tonight and we want to be as successful as possible.
"I didn't realise Great Britain and Canada didn't get the stick off so I was running with 20 metres to the line thinking 'hey, where's everyone at?!'."
Thompson, who won double gold in Rio last year, was afforded a rousing welcome as she safely led her Jamaican teammates to victory in the women's 4x200m.
The 24-year-old anchored her team also including Jura Levy, Shericka Jackson and Sashalee Forbes home in a championship record of 1min 29.04sec, Germany (1:30.68) snatching silver from the United States by 0.21.
"There was no pressure," said Thompson. "We came on the track and do what we do."
The US defended their women's 4x800m relay title in first final of the night, Laura Roesler and Charlene Lipsey pulling away in the final two legs after teammates Chanelle Price and Chrishuna Williams were involved in an early nip-and-tuck battle with Australia.
"I've had the pleasure of making all three teams that have won here in the Bahamas," said Price, whose opening leg of 2:01.73 was the quickest run on the night.
"I knew we had a target on our backs and that all the other teams wanted to take the crown from us. We also wanted to set the tone for the USA."
Belarus eventually edged the Australian quartet into third thanks to a strong anchor leg by Maryna Arzamasova, the 2015 world champion in Beijing.
Despite Olympic 400m champion Shaunae Miller-Uibo running a scorching first leg, the Bahamas failed to progress to the final of the women's 4x400m. France, with European indoor champion Floria Guei running anchor, also failed to qualify.
There was also no such luck for the Jamaican men's 4x100m quartet, missing Bolt and late withdrawal Asafa Powell, as the precision needed for baton passovers was highlighted in their first-round race.
Third leg runner Jevaughan Minzie bolted before Kemar Bailey-Cole could get the baton anywhere near to him, leaving anchor leg Yohan Blake, the 2011 world 100m champion and double 2012 Olympic silver medallist behind Bolt, with his head in his hands.
That leaves the Jamaicans needing to post a top-16 qualifying time for the London worlds by July 23.
Sunday's action sees the men compete in the 4x200m, 4x400m and 4x800m and the women in the 4x100m and 4x400m, while there will also be a mixed-sex 4x400m relay, handing Miller-Uibo a second chance to shine in front of her raucous home support.