OBJECTIVE: To determine and compare the rates of HIV superinfection and primary HIV infection in high-risk female sex workers (FSWs) in Kampala, Uganda. DESIGN: A retrospective analysis of individuals who participated in a clinical cohort study among high-risk FSWs in Kampala, Uganda. METHODS: Plasma samples from HIV-infected FSWs in Kampala, Uganda were examined with next-generation sequencing of the p24 and gp41 HIV genomic regions for the occurrence of superinfection. Primary HIV incidence was determined from initially HIV-uninfected FSWs from the same cohort, and incidence rate ratios were compared. RESULTS: The rate of superinfection in these women (7/85; 3.4/100 person-years) was not significantly different from the rate of primary infection in the same population (3.7/100 person-years; incidence rate ratio = 0.91, P = 0.42). Seven women also entered the study dual-infected (16.5% either dual or superinfected). The women with any presence of dual infection were more likely to report sex work as their only source of income (P = 0.05), and trended to be older and more likely to be widowed (P = 0.07). CONCLUSIONS: In this cohort of FSWs, HIV superinfection occurred at a high rate and was similar to that of primary HIV infection. These results differ from a similar study of high-risk female bar workers in Kenya that found the rate of superinfection to be significantly lower than the rate of primary HIV infection.