Background: North Tijuana, Mexico is home to many individuals at high risk for transmitting and acquiring human immunodeficiency virus (HIV). Recently, policy shifts by local government impacted how these individuals were handled by authorities. Here we examined how this affected regional HIV transmission dynamics. Methods: HIV pol sequences and associated demographic information were collected from 8 research studies enrolling persons in Tijuana and were used to infer viral transmission patterns. To evaluate the impact of recent policy changes on HIV transmission dynamics, qualitative interviews were performed on a subset of recently infected individuals. Results: Between 2004 and 2016, 288 unique HIV pol sequences were obtained from individuals in Tijuana, including 46.4% from men who have sex with men, 42.1% from individuals reporting transactional sex, and 27.8% from persons who inject drugs (some individuals had >1 risk factor). Forty-two percent of sequences linked to at least 1 other sequence, forming 37 transmission clusters. Thirty-two individuals seroconverted during the observation period, including 8 between April and July 2016. Three of these individuals were putatively linked together. Qualitative interviews suggested changes in policing led individuals to shift locations of residence and injection drug use, leading to increased risk taking (eg, sharing needles). Conclusions: Near real-time molecular epidemiologic analyses identified a cluster of linked transmissions temporally associated with policy shifts. Interviews suggested these shifts may have led to increased risk taking among individuals at high risk for HIV acquisition. With all public policy shifts, downstream impacts need to be carefully considered, as even well-intentioned policies can have major public health consequences.