In Kufa, six people were killed and 32 wounded in battles which erupted on Tuesday night between US soldiers and Shia Muslim militiamen loyal to Shia leader Muqtadar al-Sadr.
One fighter was killed and 21 wounded during fighting on Wednesday near two mosques in the centre of the city. And another five were killed and 11 wounded overnight.
In the nearby city of Najaf, one person was killed and 12 were injured when three mortar shells hit an industrial zone close to a US base, according to a medical source at the al-Hakim hospital.
The fighting broke out after Najaf Governor Adnan al-Zorfi announced a 72-hour truce in the area. But al-Sadr's office denied a new deal had been struck.
Two militiamen were also killed in Baghdad's poverty-stricken Shia neighbourhood of Sadr City, an official from al-Sadr's office there said.
"There is no truce with the Americans. If they enter the town (Sadr City), we open fire," said Kazem Jamal.
Shia House - a grouping of Shia political parties - issued a statement accusing US troops of responsibility for the renewed fighting in Najaf and Kufa.
"We blame occupation forces for the renewed clashes in Kufa and Najaf including the attack on the industrial neighbourhood this morning," it said.
Six Iraqis were killed in Kufa
during the clashes
"It is a clear violation of the truce and is an obstacle towards reaching a peaceful resolution to the crisis and contrary to the terms of Ambassador Christopher Ross' letter."
Ross, a former US ambassador to Syria and Algeria, is a troubleshooter for US occupation administrator, Paul Bremer.
He has been deeply involved in finding an exit from the two-month standoff between al-Sadr's militia and the US army.
Ross gave the green light on Tuesday, before the latest clashes, to a 72-hour truce thrashed out the night before between Shia leaders and occupation military commanders, according to Zorfi.
A truce effort last week was punctured by daily violence.
On Monday, around 20 members of al-Sadr's Mahdi army, two US soldiers and an Iraqi mother were killed in the running battles around Najaf.