Wan Azizah Wan Ismail, an Irish-trained medical doctor and mother of six, filed her nomination papers for the by-election in Anwar's constituency in the northern state of Penang, her party's communications director Fahmi Fadzil said.
It marks the second time Wan Azizah, 62, has stepped in for her husband during one of his controversial imprisonments.
She won Anwar's seat in 1999 after he was sacked as deputy prime minister in Malaysia's long-ruling government and jailed on sodomy and corruption charges widely considered politically motivated.
"It is not that I am asking for sympathy but don't we want to fight the injustice? I need your help and I need your prayers," she said in a speech Saturday quoted by Malaysian media.
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Wan Azizah, a petite woman who wears a traditional Muslim headscarf, is now president of Anwar's People's Justice Party.
She appears unlikely to replace him as opposition leader, however, with the baton expected to pass to Anwar's longtime protege Azmin Ali.
Anwar was jailed in February for five years for sodomising a former male aide.
He denies the charge, calling the case a conspiracy by Prime Minister Najib Razak's government to cripple the three-party opposition.
Najib has been condemned at home and abroad for the arrests of scores of opposition politicians and other critics over the past year on sedition and other charges.