Background: The COVID-19 pandemic had great impact on HIV care and prevention worldwide, including in Brazil. We compared HIV testing, recent infection, and annualized incidence according to the COVID-19 pandemic period among cisgender men who have sex with men (MSM) and transgender women (TGW). Setting: HIV and sexually transmitted infection testing, prevention, and treatment referral service in Rio de Janeiro, Brazil. Methods: We used Maxim HIV-1 Limiting Antigen Avidity EIA as part of a recent infection testing algorithm to identify recent HIV infection cases and estimate annualized HIV incidences in the pre- (March 2018–February 2020) and post-COVID-19 pandemic onset period (March 2020–January 2022). Multivariable logistic regression model assessed factors associated with recent HIV infection. Results: Among 3814 MSM and 776 TGW, 593 (12.9%) tested positive for HIV and 119 (2.6%) were identified as having recent infection. Percentage of recent HIV infection did not differ between the COVID-19 periods. Overall annualized HIV incidence rates were 6.0% (95% confidence interval [CI]: 4.2 to 7.7) and 6.6% (95% CI: 4.3 to 9.0) in the pre- and post-COVID-19 periods, respectively. During the post-COVID-19 period, higher incidence rates were observed among TGW (8.4% [95% CI: 2.9 to 13.9]), those aged 18–24 years (7.8% [95% CI: 4.0 to 11.7]), of Black race (7.9% [95% CI: 3.8 to 12.0]), and those with <12 years of schooling (7.8% [95% CI: 4.8 to 10.8]). Compared to the pre-COVID-19 period, incidence rates were significantly higher in the post-COVID-19 period for those aged >30 years and TGW, while being lower for those with more years of schooling. Conclusion: HIV incidence estimates remain high among MSM and TGW in Brazil, especially among the most vulnerable. The consequences of the COVID-19 pandemic on the HIV epidemic will likely persist and contribute to worsening HIV outcomes.