Background HIV cohort studies have been used to assess health outcomes and inform the care and treatment of people living with HIV disease. However, there may be similarities and differences between cohort participants and the general population from which they are drawn. Objective The objective of this analysis was to compare people living with HIV who have and have not been enrolled in the DC Cohort study and assess whether participants are a representative citywide sample of people living with HIV in the District of Columbia (DC). Methods Data from the DC Health (DCDOH) HIV surveillance system and the DC Cohort study were matched to identify people living with HIV who were DC residents and had consented for the study by the end of 2016. Analysis was performed to identify differences between DC Cohort and noncohort participants by demographics and comorbid conditions. HIV disease stage, receipt of care, and viral suppression were evaluated. Adjusted logistic regression assessed correlates of health outcomes between the two groups. Results There were 12,964 known people living with HIV in DC at the end of 2016, of which 40.1% were DC Cohort participants. Compared with nonparticipants, participants were less likely to be male (68.0% vs 74.9%, P<.001) but more likely to be black (82.3% vs 69.5%, P<.001) and have a heterosexual contact HIV transmission risk (30.3% vs 25.9%, P<.001). DC Cohort participants were also more likely to have ever been diagnosed with stage 3 HIV disease (59.6% vs 47.0%, P<.001), have a CD4 <200 cells/µL in 2017 (6.2% vs 4.6%, P<.001), be retained in any HIV care in 2017 (72.9% vs 59.4%, P<.001), and be virally suppressed in 2017. After adjusting for demographics, DC Cohort participants were significantly more likely to have received care in 2017 (adjusted odds ratio 1.8, 95% CI 1.70-2.00) and to have ever been virally suppressed (adjusted odds ratio 1.3, 95% CI 1.20-1.40). Conclusions These data have important implications when assessing the representativeness of patients enrolled in clinic-based cohorts compared with the DC-area general HIV population. As participants continue to enroll in the DC Cohort study, ongoing assessment of representativeness will be required.