Speaking in Tel Aviv on Friday during a visit to Israel, James Mattis said that in recent days the Syrian air force has dispersed its combat aircraft.
The implication is that Syria may be concerned about additional US air strikes following the cruise missile attack earlier this month in retaliation for alleged Syrian use of sarin gas.
"There can be no doubt in the international community's mind that Syria has retained chemical weapons in violation of its agreement and its statement that it had removed them all," Mattis said.
The US defence secretary said he did not want to elaborate on the amounts Syria possesses in order to avoid revealing intelligence sources.
"I can say authoritatively they have retained some, it's a violation of the United Nations Security Council resolutions and it's going to have to be taken up diplomatically and they would be ill advised to try to use any again, we made that very clear with our strike," said Mattis.
Israeli defence officials said this week that Syria still has up to three tonnes of chemical weapons in its possession.
It was the first specific intelligence assessment of Syrian President Bashar Assad's weapons capabilities since a deadly chemical attack earlier this month.
Assad has rejected the accusation that he was behind the attack in the opposition-held town of Khan Sheikhoun in Syria's northern Idlib province, and has accused the opposition of trying to frame his government.
Russia, Assad's principal ally, has asserted that a Syrian government air strike hit a rebel chemical weapons factory, causing the disaster.
In response to the April 4 attack, the US fired 59 missiles at a Syrian airbase it said was the launching pad for the attack.
Syria agreed to give up its chemical weapons arsenal to avert US strikes following a chemical-weapons attack in opposition-held suburbs of Damascus in August 2013 that killed hundreds of people and sparked worldwide outrage.
Before that disarmament, Assad's government disclosed it had about 1,300 tonnes of chemical weapons, including sarin, VX nerve agent and mustard gas.
The entire stockpile was said to have been dismantled and shipped out under international supervision in 2014 and destroyed.
But doubts began to emerge soon afterwards that not all such armaments or production facilities were declared and destroyed.
There also is evidence that the Islamic State of Iraq and the Levant (ISIL) group and other armed anti-government groups have acquired chemical weapons.
The Syrian government has been locked in a six-year civil war against an array of opposition forces. The fighting has killed an estimated 400,000 people and displaced half of Syria's population.
The UN says 4.72 million Syrians are in hard-to-reach areas, including 600,000 people under siege, mostly by the Syrian army, but also by rebels or ISIL.
There has been a series of evacuations in recent months, mostly around the capital Damascus but also from the last rebel-held district of Syria's third city Homs.
The transfer of hundreds of Syrian evacuees resumed on Friday after being blocked for 48 hours at Rashidin transit point where a suicide car bombing killed 126 people, 68 of them children, on Saturday.
A total of 3,000 evacuees had left their homes in Fouaa and Kefraya, two besieged government-held towns, at dawn on Wednesday as part of a deal under which residents and fighters are also being evacuated from the rebel-held areas surrounded by government forces.
But the evacuees were forced to spend two nights in their buses at the marshalling area after last-minute disagreement over the release of prisoners held by Assad's government.
As the transfer resumed on Friday it was unclear if the prisoners had been freed.
Ten of the 45 buses carrying civilians and fighters from Fouaa and Kefraya left the marshalling area in rebel-held Rashidin, the British-based Syrian Observatory for Human Rights (SOHR) said.
The buses from Fouaa and Kefraya entered Syria's second city Aleppo, under full government control since December, Rami Abdel Rahman, SOHR chief, told AFP news agency.
Dozens of armed fighters were guarding the buses at Rashidin for fear of another attack.
Meanwhile, all of the 11 buses evacuating civilians and fighters from Zabadani and two other besieged rebel-held areas around Damascus were also on the move, according to the SOHR.
The buses from Zabadani and surrounding areas headed for rebel-held Idlib province in the north-west.