The strikes, ordered by President Joe Biden, were in response to militia drone attacks on US personnel and facilities in Iraq.
The US military said in a statement it was targeting operational and weapons storage facilities at two locations in Syria and one location in Iraq.
He did not disclose whether he believed anyone had been killed or injured, but officials said assessments were underway.
Iraqi militias, aligned with Iran, have named four members of the Kataib Sayyed al-Shuhada faction who they say were killed in the attack on the Syrian-Iraqi border.
The militia vowed to retaliate.
The U.S. strikes mark the second time Biden has ordered retaliatory strikes against Iranian-backed militias since taking office five months ago.
He last ordered limited strikes in Syria in February, this time in response to rocket attacks in Iraq.
“As tonight’s strikes demonstrated, President Biden has made it clear that he will act to protect US personnel,” the Pentagon said in a statement.
The strikes came even as the Biden administration seeks to potentially revive a 2015 nuclear deal with Iran.
The decision to retaliate appears to show how Mr Biden aims to compartmentalize such defensive strikes, while simultaneously engaging Tehran in diplomacy.
Critics of Mr Biden say Iran cannot be trusted and point to the drone attacks as further proof that Tehran and its proxies will never accept a US military presence in Iraq or Syria.
The president and the White House declined to comment on the strikes on Sunday.
Mr Biden will meet Reuven Rivlin, the outgoing president of Israel, at the White House on Monday for a broad discussion that will include efforts by Iran and the United States to reinstate the Iran nuclear deal.
These efforts have raised serious concerns in Israel, Iran’s big rival.
U.S. officials believe Iran is behind an escalation of increasingly sophisticated drone attacks and periodic rocket attacks against its personnel and facilities in Iraq, where the U.S. military has helped Baghdad to fight the remnants of the Islamic State.
Two US officials, speaking to Reuters on condition of anonymity, said Iran-backed militias carried out at least five drone attacks on facilities used by US and coalition personnel in Iraq since April.
Pentagon says targeted facilities are used by Iranian-backed militias including Kataib Hezbollah and Kataib Sayyid
One of the targeted facilities was used to launch and retrieve the drones, a defense official said.
The US military carried out strikes with F-15 and F-16 planes, officials said, adding that the pilots had returned from the mission safely.
One of the officials told Reuters: “We assess that each strike has hit the intended targets. “
The Iraqi government is struggling to cope with militias ideologically aligned with Iran who are accused of rocket attacks against US forces and involvement in the murder of peaceful pro-democracy activists.
Earlier in June, Iraq released Iran-aligned militia commander Qasim Muslih, who was arrested in May on terrorism-related charges, after authorities found insufficient evidence against him.