The objective of this study was to measure HIV prevalence and risk behaviour in injecting drug users (IDUs), male sex workers (MSWs), Hijras (transgenders), female sex workers (FSWs) and male truckers in Karachi and Lahore, Pakistan. The design was a linked-anonymous cross-sectional study of individuals identified at key venues or through peer referral. Approximately 400 respondents in each group (200 for Hijras) responded to a standardized questionnaire and were tested for HIV antibodies at each site. In Karachi, 23% of IDUs and 4% of MSWs were HIV positive, and HIV-positive individuals were identified in all risk groups in at least one city. Two-thirds of all IDUs used a shared needle in the previous week, and unprotected commercial sex activity with men and women was high. The HIV epidemic has entered IDU and male and female commercial sex networks in Karachi and Lahore. Targeted intervention services must be scaled up and risk group surveillance intensified.