Some 1,425 delegates from across the country began casting their votes by secret ballot early in the morning with all eyes on Mmusi Maimane, 34, the party's parliamentary leader and the favourite to succeed outgoing leader Helen Zille, who has led the party for eight years.
Maimane was raised in the township of Soweto - heartland of the anti-apartheid struggle - and joined the Democratic Alliance (DA) only in 2009.
"Voting has closed and counting is now underway. Results are expected by 1 pm (1630 IST)," said Congress Presiding Officer Penny Tainton.
Today's vote marks "a milestone for the DA and South African politics", according to an editorial in the Sunday Times.
"For much of the past two decades, our political contest has been a black versus white affair, with the ANC (African National Congress) seen as the party of the previously oppressed and the DA as a party of white interests," the newspaper added.
Many delegates would not reveal who they were voting for, although Lydia Makoe from Limpopo province in the north made her preference for Maimane clear.
"He is our new leader, there can't be any better candidate," she said.
In 2014 Maimane was elected as the party leader in parliament, with Zille's backing, and he has on several times locked horns with the ruling ANC lawmakers, including President Jacob Zuma.
In 2014 he also unsuccessfully ran for position of premier of Gauteng province, South Africa's economic hub which includes the city of Johannesburg.
She will announce her successor.
Under Zille the DA made inroads in areas long dominated by the ANC, and it is looking to grow its support in the next local government elections in 2016.