Mattis's warning came amid reports President Bashar al-Assad's forces have used chlorine gas in the rebel-held area near Damascus, and he underscored his remarks by referencing President Donald Trump's strike on a Syrian airbase after an alleged chemical attack last year.
"We have made it very clear that it would be very unwise to use gas against people, civilians on any battlefield," Mattis told reporters accompanying him on a trip to Oman.
"I just want to reiterate that it would be very unwise for them to use weaponised gas, and I think President Trump made that very clear early in his administration."
Mattis said he was aware of "an awful lot of reports about chlorine gas use or about symptoms that could be resulting from chlorine gas," but indicated he did not have conclusive evidence.
Trump last April ordered a missile strike against a regime airbase at Shayrat, after Washington said it used the facility to launch a sarin nerve gas attack on the rebel-held town of Khan Sheikhun, killing scores of civilians.
The use of chlorine as a weapon is banned under international law and Russia was supposed to oversee the destruction of Syria's chemical weapons arsenal.
The fact that Assad may still have chemical weapons shows "either Russia is incompetent or in cahoots with Assad," Mattis said.
Syria's besieged Eastern Ghouta region is the last opposition-controlled pocket near Damascus. For nearly three weeks, regime forces have pounded it in an assault that has killed over 1,000 civilians.
The Pentagon chief also took aim at Russia, which has been propping up Assad's regime since 2015 and helping in anti-rebel operations.
Assad "could not be in power absent Russia's unfortunate veto in the UN years ago and the Russians' full-throated military support for Assad," he said.
Mattis declined to say whether the use of gas would represent some sort of trigger that would prompt a US military response.
"The president has full political manoeuver room to take the decision that he believes appropriate," he said.
"There are other Western nations that have been in contact with us that are watching this very closely and are completely aligned with us and what I just said -- that it would be very unwise for someone to use gas.
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