Measles, despite being vaccine preventable is still a major public health problem in many developing countries. We estimated the proportion of measles susceptible children in Karachi, the largest metropolitan city of Pakistan, one year after the nationwide measles supplementary immunization activity (SIA) of 2007–2008. Oral fluid specimens of 504 randomly selected children from Karachi, aged 12–59 months were collected to detect measles IgG antibodies. Measles antibodies were detected in only 55% children. The proportion of children whose families reported receiving a single or two doses of measles vaccine were 78% and 12% respectively. Only 3% of parents reported that their child received measles vaccine through the SIA. Among the reported single dose measles vaccine recipients, 58% had serologic immunity against measles while among the reported two dose measles vaccine recipients, 64% had evidence of measles immunity. Urgent strengthening of routine immunization services and high quality mass vaccination campaigns against measles are recommended to achieve measles elimination in Pakistan.