Former world 100 metres record holder Asafa Powell and Olympic 4×100 metres relay silver medallist Sherone Simpson tested positive for the same banned stimulant, oxilofrine.
Powell, 30, was the last man to hold the individual 100m record before countryman Usain Bolt broke it in 2008.
Powell later helped Jamaica to win 400m relay gold at the 2008 Olympics in Beijing.
He has run 9.88 seconds this year for the 100m, but failed to make the Jamaican team for next month’s World Championships.
Powell, who has been in good form recently said he had not wilfully taken supplements or substances that broke any rules.
“I am not now, nor have I ever been, a cheat,” he said in a statement.
Simpson, 28, who finished equal second in the 100 metres at the 2008 Beijing Games and won a gold medal in the 2004 Athens 4×100 metres relay, also denied knowingly taking a banned substance.
“This is a very difficult time for me,” she said in a statement.
“As an athlete, I know I am responsible for whatever goes into my body. I would not intentionally take an illegal substance of any form into my system.”
Oxilophrine has similar properties to ephedrine, although it has a different chemical structure, and both are on the World Anti-Doping Agency banned list.
According to the country’s anti-doping authority, Powell and Simpson are among five athletes, who have tested positive for banned performance-enhancing drugs at last month’s Jamaican championships.
Herb Elliott, chairman of the Jamaican Anti-Doping Commission (JADCO), confirmed the body had received “reports of adverse analytical findings from ‘A’ samples.”
“We cannot disclose any further information until the athletes have responded to notification of the ‘A’ sample.”
The managers for Bolt and world 100 metres champion Yohan Blake said their athletes were not involved. Blake did not compete at the championships because of injury while Bolt won the 100 metres.
Powell and Simpson’s doping positives come a month after Jamaican Olympic champion Veronica Campbell-Brown tested positive for a banned diuretic.
Tyson Gay fails test
American former world champion Tyson Gay has also failed drug tests.
Gay, 30, was told by the US Anti-Doping Agency on Friday that his A sample from an out-of-competition test in May was positive.
Gay, who is the fastest man in 2013 and won gold in the 100m, 200m and 4x100m at the 2007 World Championships, is waiting for the results of his B sample.
He has already withdrawn from next month’s World Championships in Moscow.
“I don’t have a sabotage story… I basically put my trust in someone and was let down,” he said.
“I know exactly what went on, but I can’t discuss it right now.
“I hope I am able to run again, but I will take whatever punishment I get like a man.”
Dope busts have particularly afflicted the 100 metres with eight of the fastest 14 men ever having served, or about to serve drug, suspensions.
Turks under scrutiny
The Jamaican and American athletes aren’t the only ones causing a stir as media reports said that up to 30 Turkish athletes faced doping bans after the International Association of Athletics Federations confirmed the country had been targeted over concerns about abnormal biological passport values.
Biological passports track changes in athletes’ blood profiles, which could be caused by doping.