Southern Sudan leader Salva Kiir has made his strongest call for full independence when the region's status is decided at a referendum due in 2011, BBC reported.
He said voting for unity with northern Sudan would make southerners "second class citizens" in their own land.
A referendum in the now semi-autonomous oil-rich south was part of the 2005 deal that ended decades of civil war.
A BBC correspondent in Sudan says Mr Kiir's comments are likely to add to tensions between the north and south.
Previously officials have been careful in public to at least promote the unity between north and south, as the peace deal stipulates, says the BBC's Peter Martell in the South Sudan capital Juba.
Salva Kiir was speaking at a special church service to pray for peace, timed to mark the start of voter registration for multi-party elections due in April 2010 and the January 2011 referendum.
"When you reach your ballot boxes the choice is yours. You want to vote for unity, so that you become a second-class (citizen) in your own country, that is your choice," he told the congregation at St Teresa's Catholic Cathedral in Juba.
"You would want to vote for independence, so that you are a free person, in your independent state, that will be your own choice. And we will respect the choice of the people."
In October, South Sudan said it had achieved a breakthrough in talks with the north over terms for the referendum.
Vice-President Riek Machar said the vote would require a simple majority as long as two-thirds of those eligible took part.
In the past, the Khartoum government had insisted that 75% of voters must agree to independence.
Mr Machar said all southerners would be allowed to vote, including those in Khartoum and those outside Sudan.