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Earlier this month, Gay revealed he had tested positive in an out-of-competition test he took on May 16.
Three people familiar with the case confirmed that Gay had multiple positives this year. One of those people said one positive came at nationals and the US Anti-Doping Agency has notified him of that result.
One person familiar with the case said that multiple positives over a short period of time are a sign of an athlete who wasn't trying to hide anything, but simply didn't know he was taking a banned drug.
The people did not want their names used because the doping case against Gay is ongoing and the details have not been made public.
Gay's representatives did not immediately respond to attempts to reach them.
The sprinter has already surrendered his spot at next month's world championships. If a positive test from nationals is confirmed by his 'B' sample, those results would be vacated, though it's likely they would be anyway because of his May 16 positive.
The May 16 test has been confirmed by a test of the 'B' sample, one of the people familiar with the case said.
A first-time offence for doping usually brings with it a two-year ban, though athletes who co-operate with USADA sometimes get reduced penalties.
Gay is co-operating with USADA.
"The additional positive sample is consistent with him taking responsibility," USADA CEO Travis Tygart said.
"He should be commended for that and for removing himself from the world championships, which we all should appreciate. The sample was expected, as he works with us for the fair resolution based on the rules, given the fact of his case."
When Gay admitted to the earlier positive in a phone interview nearly two weeks ago, he fought back tears.
"I don't have a sabotage story. I don't have any lies. I don't have anything to say to make this seem like it was a mistake or it was on USADA's hands, someone playing games," he said.
"I don't have any of those stories. I basically put my trust in someone and I was let down."
Since news of his positive, media reports have linked Gay to Clayton Gibson, an anti-aging doctor based in Atlanta. In an email sent to the Associated Press, Gibson would not confirm Gay was among his patients.
Gay has been the most-watched US male sprinter for the past several years and has long sold himself as a clean athlete.
The 30-year-old, who won the world championship in the 100m, 200m and 4x100m relay in 2007, took part in USADA's "My Victory" programme - in which athletes volunteer for enhanced testing to prove they're clean - and his results never raised red flags.
Until, that is, the out-of-competition test in May. Results came back positive for a banned substance, the identity of which he has not been revealed.
The latest news adds to a slew of negative headlines for athletics. Jamaican Olympic champion, Veronica Campbell-Brown, tested positive for a banned diuretic at a meet on the island in May.
A month later, Asafa Powell, the former 100m world record holder, tested positive for a stimulant at Jamaica's national championships, as did his team-mate, Sherone Simpson, a three-time Olympic medalist.
In a recent statement, Powell said he had "retained legal counsel" and couldn't "discuss all the details of the case."
"I look forward to demonstrating to everyone that I am a clean athlete. I always have been, and I always will be," Powell said.
"I look forward to donning my country's national (colors) and as I step into the blocks at the starting line I will remain, as always, proud to be Jamaican."
Gay, finally feeling healthy after years of nagging injuries, was among those expected to push Usain Bolt at worlds next month, but instead of racing in Moscow, he'll be resolving his doping case.
Asked on Thursday about the series of doping cases, Bolt said, "definitely, it's going to set us back a little bit."
He insisted he was clean.
"So I'm just going to continue running, using my talent and just trying to improve the sport," he said.
