Singh, 73, and one of his longtime associates were killed early this morning when the car they were traveling in rammed into a lorry at the North South Expressway in Kampar town in Perak state.
His son Gobind Singh Deo confirmed that his father had died on the spot. His assistant Michael Cornandez was also killed while two others, his son Ram Singh and an Indonesian maid, were seriously injured.
Singh, a tireless defender of human rights activists, was on his way to Penang to attend a court case later in the day.
Born in Penang to an Indian Punjabi immigrant father, Singh studied law at the University of Singapore and was regarded as Malaysia's top criminal lawyer.
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Singh was suspended from parliament numerous times, charged for sedition, and was detained under Malaysia's internal security laws for an year.
His reputation as a lawyer and opposition politician had earned him the nickname "the Tiger of Jelutong".
Jelutong is a town in Penang.
Singh's political career began in 1970 when he joined the DAP. He won a seat in the Kedah State Legislative Assembly in 1974. He was first elected to parliament in 1978 as the representative for Jelutong, Penang, and held the seat for more than 20 years until losing it in 1999.
A car accident in 2005 had left Singh partially paralyzed and dependent on a wheelchair. Despite this, he continued his legal and political careers.
Prime Minister Najib Razak, who is currently on an official visit to Turkey, tweeted from Ankara to extend his condolences to the politician's family members.
"I am shocked to hear of Karpal Singh's tragic passing," Najib said in a statement issued by his office here.
"In politics, he was an implacable leader; in law, a committed advocate," Najib said.
Opposition leader Anwar said the news came "like a lightning bolt".
He called Karpal "an inspiring symbol for the struggle against oppression and injustice".