The seroprevalence, risk factors and genotypes of hepatitis C virus (HCV) in groups with high-risk sexual behavior (persons with multiple sexual partners, men who have sex with men, commercial sex workers and their clients and persons with sexually transmitted diseases) in seven Croatian cities were analyzed. A total of 821 participants without history of injecting drug use were included in the study. Anti-HCV prevalence among risk groups varied from 2.9% to 8.5% with an overall prevalence of 4.6% (95% CI = 3.2–6.1) compared with 0.5% (95% CI = 0.0–1.5) in controls (pregnant females; OR = 9.66; 95% CI = 1.32–70.7). HCV-RNA was detected in 73.1% anti-HCV positive patients. Three of the seronegative cases (2.1%) were also found to be HCV-RNA positive (“window period”). Genotype 1 was most commonly detected (55.6%). The most prevalent subtypes were 1a (38.9%) and 3a (38.9%). Sociodemographic characteristics (age, gender, marital status and level of education) were not associated with anti-HCV seropositivity. Among sexually transmitted disease markers, a higher seroprevalence of HCV infection was found in subjects with a history of HBV infection (10.5% vs. 3.8%, P = 0.002) and gonorrhea (13.2% vs. 4.2%, P = 0.011). No other factors reflecting risk sexual behavior such as sexual orientation, number of sexual partners and number of risk behaviors were associated with HCV seroprevalence. J. Med. Virol. 81:1348–1353, 2009. © 2009 Wiley-Liss, Inc.