Yemen has said its security forces carried out the raid in the southern province of Abyan last December.
Amnesty released photographs, which it said were apparently taken after the attack, of what it said was a US-made cruise missile carrying cluster munitions.
The photographs appear to show damaged parts of a Tomahawk cruise missile, which Amnesty said is a weapon held only by US forces, and of a cluster bomblet.
"The US government must disclose what role it played in the ... attack, and all governments involved must show what steps they took to prevent unnecessary deaths and injuries," Philip Luther, deputy director of Amnesty International's Middle East and North Africa program, said.
"A military strike of this kind against alleged militants without an attempt to detain them is at the very least unlawful," he said in a statement.
In Washington, a Pentagon spokesman declined to comment on the strike, saying questions on operations against Al-Qaeda should be posed to the Sanaa government.
"That said, the Yemen government should be commended for dealing with the Al-Qaeda threat in their nation. Al Qaeda in the Arabian Peninsula threatens the stability of the region and poses an increasing threat to Yemenis and Americans," Pentagon spokesman Bryan Whitman said.
"We strongly support actions against Al-Qaeda in the Arabian Peninsula and cooperate closely with Yemen and other countries on counterterrorism initiatives," he added.
The Yemeni government expressed regret in March over civilian deaths in the attack and said it would compensate families of civilian victims.
Amnesty said it was very worried by the evidence that cluster bombs had been used. Neither the United States nor Yemen has signed the Convention on Cluster Munitions, a treaty to ban such weapons which is due to take effect in August.
"The fact that so many of the victims were actually women and children indicates that the attack was in fact grossly irresponsible, particularly given the likely use of cluster munitions," Luther said.
Yemen said on Dec. 17 that its security forces backed by warplanes had killed up to 30 Al-Qaeda militants.
Residents and opposition groups said about 50 civilians were killed, including women and children. Amnesty said the attack killed 41 local residents, including 14 women and 21 children, as well as 14 alleged members of Al-Qaeda.
The New York Times reported shortly after the attack that the United States had given military hardware, intelligence and other support to Yemeni forces involved in the raids.
Al Qaeda's wing in Yemen, said the failed Christmas Day bombing of a US-bound plane, for which it claimed responsibility, had been in retaliation for the Dec. 17 raid which it accused the US Air Force of carrying out.
Amnesty said it obtained the photographs from a confidential source during a visit to Yemen in March. Due to security restrictions, Amnesty said it had not yet been able to visit the site to verify the images.
