Pakistani protestors burn an American flag with pictures of Pakistan's President General Pervez Musharraf
At least 2,000 people shouted anti-American slogans in Islamabad while hundreds of police maintained order.
Similar demonstrations, all peaceful, were held in major cities throughout Pakistan.
Protesters like Ejaz Afzal Khan demanded those involved in the alleged desecration of the Koran be held accountable.
"They should be charged for that and they should be punished,” he said. “They should be handed over to the international court or to Muslims to punish them."
The marches were organized by fundamentalist Islamic groups opposed to Pakistan's support for the U.S.-led war against terrorism.
Abdul Rashid Gharzi says he joined the march in Islamabad to protest Pakistan's president, Pervez Musharraf.
"We feel it is because of Pervez Musharraf, who is serving as an agent of the United Sates. He is, in fact, responsible for all the dishonor to our holy book as well as our country,” he added.
Earlier this month the U.S. magazine Newsweek reported an incident where a copy of the Koran was thrown in a toilet during an interrogation.
However the magazine quickly retracted the story and a subsequent military investigation found no evidence supporting the specific allegation.
Officials did confirm five other incidents where the Koran was "mishandled" at Guantanamo Bay.
The U.S. State Department says any cases of religious abuse are contrary to U.S. policy.
Meanwhile, security officials in Pakistan remain on high alert after a suicide bomb attack at a Muslim shrine in Islamabad.
The attack is reportedly linked to sectarian conflicts between militant members of Pakistan's Sunni majority and its Shi'ite minority.