Chambers ran 10.52 seconds to finish four-tenths of a second behind winner Keston Bledman of Trinidad and Tobago in an international competition at Ponce, Puerto Rico.
He was sixth in his heat and eighth overall in the combined results for two heats.
British rival Tyrone Edgar was among those who finished ahead of former world indoor champion Chambers. Edgar clocked 10.42 for fourth.
The race was Chambers' first since the Court of Arbitration for Sport ruled last month against the British Olympic Association's lifetime Olympic ban for drug cheats, clearing the way for the sprinter and cyclist David Millar, who have both served bans for doping, to be considered for selection to Britain's Olympic team.
Chambers begins his European season with a race over 150 metres against American world silver medallist Walter Dix in Manchester, England on May 20. He has been training in Jamaica with former world record holder Asafa Powell.
Puerto Rican world silver medallist Javier Culson delighted home fans with the year's fastest 400 metres hurdles.
Culson sped home in 48.00 seconds to defeat Trinidad and Tobago's Jehue Gordon and American Bershawn Jackson.
Former US. collegiate high hurdles champion Ryan Wilson produced the biggest surprise, out-racing both US world champion Jason Richardson and Cuban world record holder Dayron Robles.
Wilson edged out Richardson by one thousandth of a second to win the 110 hurdles in 13.424 seconds.
Robles, who is scheduled to compete in the United States next month for the first time, was third in 13.47 seconds.
British Olympic hopeful and world indoor silver medallist Tiffany Porter easily won the women's 100 hurdles as she ran 12.79 seconds to defeat U.S. indoor champion Kristi Castlin.
American Tianna Madison swept the women's sprints, winning the 100 in 11.01 seconds and the 200 in 22.37, the year's second fastest time.