"That would be a mistake," Mattis told reporters at the Pentagon.
Assad and rebel Kurdish forces have recently exchanged angry words, stoking fears that fighting might break out between the two camps.
There is currently a demarcation line between areas controlled by the US-led coalition battling jihadists with the Islamic State group, mainly in eastern Syria, and those controlled by Syria and its Russian allies in western Syria.
"We have said that we would operate on one side and the Russians on the other," Mattis said. "And we are still taking (the IS) down. Nothing has changed."
Assad had criticised the semi-autonomous Kurds in the past, but his mid-December comments were harsher than any since the Syrian unrest broke out in 2011.
"When we talk about those referred to as 'the Kurds', they are in fact not just Kurds," Assad said in remarks released by the Syrian presidency.
"All those who work for a foreign country, mainly those under American command... are traitors."
The comments prompted an angry response from the Kurdish-dominated Syrian Democratic Force (SDF), which seized swaths of northern and eastern Syria from the IS in a months- long assault backed by US-led coalition air support.
They accused the regime of freeing "terrorists" from its prisons so they could "shed the blood of Syrians of all stripes."
In early December a Pentagon spokesman said that US forces would remain in Syria "as long as necessary" -- and on Friday Mattis added that US diplomats will soon be on the ground in eastern Syria to organize de-mining and reconstruction projects.
"The military would move our diplomats around, make certain to protect them," Mattis said.
Mattis did not say what legal basis will be used to send in US diplomats to Syria, as the US-led coalition is authorised under Article 51 of the United Nations charter only to battle IS jihadists.
The Assad regime controls about 55 per cent of Syria after defeating rebels and jihadists with Russian support. Kurdish forces control 28 per cent of the country.
When asked if the ultimate US objective was to partition Syria, Mattis's response was succinct: "No.
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