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Abstract Background There is little evidence on the need for differentiated HIV prevention services for men who have sex with men (MSM), female sex workers (FSW) and people who inject drugs (PWID in Nigeria. The aim of the study was to determine and compare the HIV sexual risk profiles of FSW, MSM and PWID resident in Nigeria; and identify factors associated with condom use among the groups. This will help identify if differentiated HIV prevention services are needed for MSM, FSW and PWID in Nigeria. Methods This is a cross-sectional study. Data on sexual practices (anal, vaginal and oral sex), history of alcohol and psychoactive substance use, and high risk sexual behaviors for HIV infection (inconsistent use of condom) was collected from study FSW, MSM and PWID resident in Enugu, Nassarawa, Benue, and Akwa-Ibom States of Nigeria between April and June, 2015. Association between sexual practices, alcohol and psychoactive substance use, and HIV sexual risk behaviors; and differences in sexual risk behaviors of MSM, FSW and PWID were determined using Pearson chi-square for categorical variables, and t-test for continuous variables. Determinants of condom use in the last 30 days were identified using logistic regression analysis. Results The study population consisted of 188 (38.5%) FSW, 145 (29.7%) MSM and 155 (31.8%) PWID. MSM (AOR: 0.17; 95%CI: 0.05–0.67; p = 0.01) and PWID (AOR: 0.07; 95%CI: 0.02–0.21; p