Chiefs or representatives of 61 tribes from across the North African country called for an end to Qadhafi's four-decade rule, in a joint statement released by French writer Bernard-Henri Levy.
"Faced with the threats weighing on the unity of our country, faced with the manoeuvres and propaganda of the dictator and his family, we solemnly declare: Nothing will divide us," said the statement, released on Wednesday in Benghazi.
"We share the same ideal of a free, democratic and united Libya.
"The Libya of tomorrow, once the dictator has gone, will be a united Libya, with Tripoli as its capital and where we will at last be free to build a civil society according to our own wishes," it said.
Levy has become an unofficial spokesman in Paris for the revolt and is credited with pressing President Nicolas Sarkozy to mobilise international political and military support for it.
"Each of the tribes in Libya is represented by at least a representative. In this list of 61 signatures, some tribes are represented 100 percent, others are still divided," he said.
Their call came as rebels said they had managed to push back Qadhafi's forces and secure the port of besieged Misrata, a day after it came under sustained rocket fire.
As the United States and its allies pondered how to help the rebels, US President Barack Obama formally ordered a drawdown of $25 million (17 million euros) in urgent, non-lethal aid to the Transitional National Council.
UN Secretary General Ban Ki-moon said Kadhafi's regime "has lost both legitimacy and credibility, particularly in terms of protecting its people and addressing their legitimate aspirations for change."