Southern leaders had urged voters to turn out en masse on the first day. A 2005 peace deal requires a turnout of at least 60 percent for the referendum to be valid. The outcome will then be decided by simple majority.
A few voters wore their opinions on their chests with T-shirts demanding independence.
Most donned the Western suits and brightly coloured floral dresses normally reserved for church in this largely Christian region, but they showed no less enthusiasm for separation from the Muslim, mainly Arab north.
While the first day of voting was mostly peaceful, an official said clashes between renegade militiamen and south Sudanese troops disrupted voting in part of the key oil Unity state, which abuts the north-south border.
"There has been some fighting because of certain militiamen but I am assured that the situation has been contained," said the organising commission number two, Chan Reec.
Reec, who had earlier been effusive about the massive turnout, later appealed for more consideration to be shown to women and the elderly at polling stations.
Polls had been scheduled to close at 5:00 pm (1400 GMT), but many polling stations visited by AFP in the regional capital Juba stayed open for another two hours or more to deal with the huge backlog of eager voters.
Some 3.75 million people are registered to vote in the south and around 117,000 in north Sudan, the majority in the capital Khartoum. Emigres were also able to vote in eight countries abroad.
Results are not expected until early next month because of the problems involved in collecting ballot boxes in a vast, war-ravaged region which has just 40 kilometres (25 miles) of tarmac road.
The independence referendum is the centrepiece of the 2005 north-south peace deal that ended a devastating 22-year civil war in which some two million people were killed and another four million fled their homes.
South Sudanese president Salva Kiir was among the first to cast his ballot in Juba.
"This is the historic moment the people of south Sudan have been waiting for," Kiir said, holding up his hand to reporters to show the indelible ink that demonstrated he had voted.
US President Barack Obama, who aides said had voiced a deep personal commitment to ending the north-south conflict in Sudan, hailed the successful first day of voting and pledged Washington's continued support.
"I am extremely pleased that polling has started for the southern Sudan referendum, and congratulate the people of southern Sudan who are determining their own destiny," he said.
"The United States will remain fully committed to helping the parties solve critical post-referendum issues regardless of the outcome of the vote," Obama added.
US envoys Scott Gration and John Kerry as well as Hollywood star George Clooney watched as Kiir voted at a polling station set up at the memorial to late rebel leader John Garang in Juba.
It was Garang who signed the 2005 accord that provided for Sunday's referendum, shortly before his death in a mysterious helicopter crash on his way back from Uganda.
The rebel leader's widow Rebecca said: "I have mixed feelings about this day for I know that my husband did not die in vain and I know that freedom has a price."
After touring a polling station with ex-US president Jimmy Carter, former UN chief Kofi Annan said: "It is important that the energy and enthusiasm lead to solid results that are accepted by everybody."
Britain, Norway and the United States, the three main Western brokers of Sudan's north-south peace process, welcomed President Omar al-Bashir's pledge to respect the result of the vote, which they termed a "historic step."
"We are encouraged by the strong public commitments of both Presidents Bashir and Kiir to continue negotiations on post-referendum issues and to foster cooperation between the north and south," they said.
Bashir, an army man who led the north's war effort against the south for a decade and a half before signing the peace deal six years ago, has said he will respect the outcome of the vote if it is "free and transparent."