Background: The national HIV scenario in India has improved impressively during past two decades. Contrastingly, the State-level HIV sentinel surveil-lance and routine programme data from various testing facilities indicated an increasing HIV prevalence among antenatal clinic attendees and female sex workers (FSWs) in three districts of Assam. Objectives: 1) To explore the reasons behind increasing adult HIV prevalence and 2) To understand the role of FSW and Migrants/long distance truckers as drivers of the HIV epi-demic in these districts. Methodology: Three sources of data were utilized in this concurrent mixed methods study—1) existing programme data for past three years; 2) questionnaire-based data for a case-control study, where dur-ing past six months at designated testing facilities HIV(+) married women were “cases” and age-matched HIV(?) women from the same district were “controls”. Spouses of these women were also interviewed separately; and 3) a qualitative study, where focus group discussions were conducted among FSWs, outgoing male migrant labourers and long-distance truckers. Results: The study revealed high levels of unsafe sexual practices among the FSWs. Sometimes in poverty-struck areas, women, not identifying themselves as sex workers, sold sex on a part-time basis to earn extra money for financial sup-port and often remained invisible to the programme. The clients of the sex workers, male migrants and truckers also revealed various risk behaviours for contracting and transmitting HIV infection. Conclusion: Population and context specific programme strategies are required to halt the rising trend of HIV infection in the general population in Assam.