Nasreen was shot dead by Ilyas Khan and his son Qasim in Karachi's Orangi town area as he suspected she was having an affair with a man, while her husband was working in Saudi Arabia.
A senior police official said that Ilyas and his son Qasim were arrested for the honour killing.
"Illyas with the help of his son shot dead his sister-in- law, Nasreen, who he suspected was having an affair with a man as her husband was working in Saudi Arabia," Senior Superintendent Police Akhter Farooq said.
"Moments after seeing him, they fired at him. He is under treatment at the Abbasi Shaheed hospital," he said.
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Killing of women relatives in the name of honour is a menace still prevalent in many parts of Pakistan.
The menace of honour killing has been a major problem in the low lying slum areas of Karachi particularly Orangi town where a large population of Bengalis, Pushtuns, Urdu speaking Muhajirs and Punjabis reside.
In April, police arrested a young man in Orangi Town for slitting his teenage sister's throat and watching her bleed to death.
In March this year a man shot dead both his sisters in Sahiwal district in Punjab after suspecting them of having loose character.
The two sisters were killed just a few days after Pakistani documentary filmmaker, Sharmeen Obaid Chinoy, won an Oscar for her film, "A Girl in the River: The Price of Forgiveness", which revolved around honour killings.
Pakistan amended its criminal code in 2005 to prevent men who kill female relatives escaping punishment by pardoning themselves as an "heir" of the victim.