Mr Obama said in a statement that “After 50 years of civil wars that have killed two million people and turned millions more into refugees this is the opportunity before the people of Southern Sudan.”
He added that the action of Sudanese leaders would help determine whether Sudanese people move “toward peace and prosperity, or slide backward into bloodshed”.
The voting started on Sunday and is expected to take a full week to give results on whether Africa’s largest country split into two or remain one.
But Sudanese President Omar al-Bashir has promised to respect the outcome, but warned an independent south would face instability. The mainly Muslim north has promised to allow the potential new country, where most people are Christian or animists, to secede peacefully.
The poll was agreed as part of the 2005 deal that ended a two-decade civil war. South Sudanese leader Salva Kiir, casting his ballot on Sunday, urged people to “be patient”, in case they were not able to vote on the first day of polling.
Southern Sudanese voters are faced with two symbols on the ballot paper – a single hand for independence or two clasped hands to remain one country.
Turnout in the referendum will be important, as the 2005 peace agreement stipulates that for the vote to be valid, 60% of the 3.8 million registered voters must take part.
Veronica De Keyes, head of the the European Union observer team in Juba, said voting appeared to have started well.
“What I observed this morning was very moving in the sense that you can feel it, in the crowd, the expectation of the people is important,” she said on Sunday.