Zahra Shahid Hussain was the senior vice-president of Pakistan’s Movement for Justice party (PTI), led by former international cricketer Imran Khan.
She was killed by gunmen on a motorcycle outside her home in the city’s upmarket Defence neighborhood.
Her murder comes on the eve of a highly-contested partial re-run of last Saturday’s general election.
The reason for the shooting is unclear.
Imran Khan has blamed the city’s dominant MQM party for her murder, a claim the party has denied.
Pakistan’s Dawn newspaper, citing police, said the shooting happened during an attempted robbery.
Our correspondent says that reports of Zahra Shahid Hussain being shot twice in the head raise suspicions that it was a targeted killing made to look like a robbery.
Local PTI leader Firdous Shamim told AFP news agency that Zahra Shahid Hussain “was leaving her home for work when three gunmen attacked her. She thought they wanted to snatch her purse and handed it over to them but they killed her”.
Zahra Shahid Hussain was reportedly rushed to hospital but succumbed to her injuries on the way.
Pakistani President Asif Ali Zardari strongly condemned the murder, describing it as a “tragic incident”.
Sunday’s partial re-run of the vote in Karachi was ordered after Imran Khan’s party accused the MQM of widespread vote-rigging and intimidation.
The MQM – which took most of the seats in Karachi – denies any irregularities.
Karachi is torn by regular violence – much of it politically motivated.
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