Tillerson demands 'Iranian-backed militias withdraw from Syria'
Washington's lead diplomat said the US was "quite concerned" by a recent confrontation that saw ally Israel bomb what it described as Iranian targets in Syria.
"This again illustrates why Iran's presence in Syria is only destabilising to the region," Tillerson said at a press conference with his Jordanian counterpart.
"We think Iran needs to withdraw its military, its militia from Syria, and allow the hope for peace process to take hold."
Longtime US foe Iran and the regional militias it backs, including Lebanon's Hizballah, have played a key role in propping up Syrian dictator Bashar al-Assad, heightening fears over Tehran's spreading influence.
Tillerson - who is on a tour of the Middle East - held closed-door talks with Syrian opposition negotiators in Amman in the wake of a January peace summit hosted by regime backer Russia.
The opposition and Kurdish groups had boycotted the congress, held just days after a ninth round of United Nations-led talks in Vienna failed to yield progress towards ending Syria's devastating conflict.
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The West views Russia's Syria peace efforts with suspicion, concerned that Moscow is seeking to sideline the UN process.
Tillerson said there were "important milestones" achieved at the conference in Russia's Black Sea resort of Sochi, most importantly a "unified commitment" to the UN efforts.
Despite Moscow insisting Syrian society would be fully represented at Sochi, almost all of the 1,400 delegates were pro-regime.
They agreed to set up a commission to re-write the country's post-war constitution.
About 150 Syrian civil society groups accused the UN of rewarding Russia by dispatching its special envoy Staffan de Mistura to the congress.
Syria's war has killed more than 340,000 people and displaced millions since it began in March 2011 with the brutal repression of anti-government protests.
Tillerson is due to meet Lebanon's political leadership in Beirut on Thursday, with the US concerned about the influence of Iran-backed Hizballah which it designates a "terrorist" organisation.
Shia movement Hizballah - the only faction to have retained its weapons after Lebanon's civil war - is a member of the Lebanese government of premier Saad Hariri.
The US has recently targeted an alleged key financier for the group with sanctions amid a probe into accusations of "narcoterrorism".
But ahead of his visit Tillerson appeared to soften Washington's tone by acknowledging "the reality that they also are part of the political process in Lebanon".