Registration centres opened at 8:00 am local time at the start of a two week process during which some five million southern Sudanese are called to add their names to the electoral list, referendum commission spokesman George Benjamin said.
In the southern capital Juba officials were awaiting the arrival of the president of the semi-autonomous region, Salva Kiir, who was expected to set an example by registering early.
Meanwhile vehicles carrying officials using loudhailers roved the streets to urge people to register en masse and to blare out music with the evocative message: "We are heading to the promised land".
"It is an historic moment for south Sudan, I am hurrying to register so I can vote in the referendum," said Gabriel Aleu, a Juba youth of about 20 years old.
Those aged 18 or over belonging to a tribe established in the south on or before January 1, 1956, the date of Sudan's independence from Britain, are eligible to vote.
However, the referendum commission has not drawn up a definitive list of southern tribes and many southerners do not have official documents proving their origins.
Voters are set to decide on 9 January whether southern Sudan wants to remain with Khartoum or become independent.
The referendum in southern Sudan and the oil-rich Abyei region straddling north and south are part of a 2005 peace deal that ended a two-decade-old civil war in Sudan which left an estimated two million dead.