INTRODUCTION: Pre-exposure prophylaxis (PrEP) programmes have been initiated in sub-Saharan Africa to prevent HIV acquisition in key populations at increased risk. However, data on PrEP uptake and retention in high-risk African communities are limited. We evaluated PrEP uptake and retention in HIV hyperendemic fishing villages and trading centres in south-central Uganda between April 2018 and March 2019. METHODS: PrEP eligibility was assessed using a national risk screening tool. Programme data were used to evaluate uptake and retention over 12 months. Multivariable modified Poisson regression estimated adjusted prevalence ratios (aPR) and 95% Confidence intervals (CIs) of uptake associated with covariates. We used Kaplan-Meier analysis to estimate retention and multivariable Cox regression to estimate adjusted relative hazards (aRH) and 95% CIs of discontinuation associated with covariates. RESULTS AND DISCUSSION: Of the 2985 HIV-negative individuals screened; 2750 (92.1 %) were eligible; of whom 2,536 (92.2%) accepted PrEP. Male (aPR = 0.91, 95% CI = 0.85 to 0.97) and female (aPR = 0.85, 95% CI = 0.77 to 0.94) fisher folk were less likely to accept compared to HIV-discordant couples. Median retention was 45.4 days for both men and women, whereas retention was higher among women (log rank, p < 0.001) overall. PrEP discontinuation was higher among female sex workers (aRH = 1.42, 95% CI = 1.09 to 1.83) and female fisher folk (aRH = 1.99, 95% CI = 1.46 to 2.72), compared to women in discordant couples. Male fisher folk (aRH = 1.37, 95% CI = 1.07 to 1.76) and male truck drivers (aRH = 1.49, 95% CI = 1.14 to 1.94) were more likely to discontinue compared to men in discordant couples. Women 30 to 34 years tended to have lower discontinuation rates compared to adolescents 15 to 19 years (RH = 0.78 [95% CI = 0.63 to 0.96]). CONCLUSIONS: PrEP uptake was high, but retention was very low especially among those at the highest risk of HIV: fisher folk, sex workers and truck drivers and adolescent girls. Research on reasons for PrEP discontinuation could help optimize retention.