BACKGROUND Key populations, including people who inject drugs (PWID), men who have sex with men (MSM), and female sex workers (FSW), are disproportionately affected by the HIV epidemic. Understanding the magnitude of, and informing the public health response to, the HIV epidemic among these populations requires accurate size estimates. However, low social visibility poses challenges to these efforts. OBJECTIVE The objective of this study was to derive population size estimates of PWID, MSM, and FSW in Kampala using capture-recapture. METHODS Between June and October 2017, unique objects were distributed to the PWID, MSM, and FSW populations in Kampala. PWID, MSM, and FSW were each sampled during 3 independent captures; unique objects were offered in captures 1 and 2. PWID, MSM, and FSW sampled during captures 2 and 3 were asked if they had received either or both of the distributed objects. All captures were completed 1 week apart. The numbers of PWID, MSM, and FSW receiving one or both objects were determined. Population size estimates were derived using the Lincoln-Petersen method for 2-source capture-recapture (PWID) and Bayesian nonparametric latent-class model for 3-source capture-recapture (MSM and FSW). RESULTS We sampled 467 PWID in capture 1 and 450 in capture 2; a total of 54 PWID were captured in both. We sampled 542, 574, and 598 MSM in captures 1, 2, and 3, respectively. There were 70 recaptures between captures 1 and 2, 103 recaptures between captures 2 and 3, and 155 recaptures between captures 1 and 3. There were 57 MSM captured in all 3 captures. We sampled 962, 965, and 1417 FSW in captures 1, 2, and 3, respectively. There were 316 recaptures between captures 1 and 2, 214 recaptures between captures 2 and 3, and 235 recaptures between captures 1 and 3. There were 109 FSW captured in all 3 rounds. The estimated number of PWID was 3892 (3090-5126), the estimated number of MSM was 14,019 (95% credible interval (CI) 4995-40,949), and the estimated number of FSW was 8848 (95% CI 6337-17,470). CONCLUSIONS Our population size estimates for PWID, MSM, and FSW in Kampala provide critical population denominator data to inform HIV prevention and treatment programs. The 3-source capture-recapture is a feasible method to advance key population size estimation.