Her return to parliament after seven years marks the second time that Wan Azizah, 62, has stepped in to lead the opposition after one of her husband's controversial imprisonments.
She faces the task of healing sharp divisions within the diverse three-party opposition alliance that Anwar has led to the brink of power but whose future is now uncertain with the removal of its central figure.
Earlier this month, Wan Azizah, a Western-trained doctor and mother of six, won a by-election for Anwar's seat.
Anwar was jailed in February for five years on charges that he sodomised a former male aide, a case he says was fabricated by the government of the Muslim-majority country to weaken the opposition.
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The prison term threatens to permanently end his political career. Anwar is now 68 years old.
Wan Azizah had earlier won the same seat in 1999, replacing Anwar as she rode a wave of public outrage over his sacking as deputy prime minister in Malaysia's long-ruling government.
Ousted in a power struggle, Anwar spent six years in jail on previous sodomy and corruption charges widely considered politically motivated.
Under the charismatic Anwar, the once-fragmented opposition has achieved unprecedented gains.
It won the popular vote in 2013 polls but the ruling coalition that has dominated Malaysia for decades held onto power.
The opposition has since been riven by internal bickering sparked by a bid launched in March by the conservative Pan-Malaysian Islamic Party (PAS) to implement harsh sharia-law punishments in a state it governs, despite an earlier pledge not to.
The penalties would include amputation for theft, and stoning to death for adultery.
Wan Azizah joins her eldest daughter with Anwar -- Nurul Izzah Anwar, 34 -- in parliament.