Background: Understanding the extent of viral hepatitis burden in specific subgroups, such as pregnant women and people living with HIV/AIDS (PLWHA), and their geographic distribution is essential for evidence-informed policy and mobilizing resources for targeted treatment and prevention efforts. However, in Cambodia, the epidemiology of hepatitis C remains uncertain. We estimated the hepatitis C virus (HCV) burden and transmission risk factors among PLWHA and pregnant women attending antenatal care (ANC) in Cambodia. Methods: Between March and April 2016, we conducted a cross-sectional survey in four diverse geographical areas: the capital city of Phnom Penh and three provinces. We collected information on demographic characteristics and risk behaviors and performed HCV antibody (Anti-HCV) testing among pregnant women attending public ANC clinics and among those receiving HIV care at the hospitals. We computed the prevalence of HCV among the two population subsets and performed logistic regression analyses to identify risk factors associated with HCV antibody positivity. Results: Of 935 participants enrolled, 510 (54.6%) were pregnant women and 425 (45.4%) were PLWHA. Anti-HCV prevalence was significantly higher in PLWHA than in pregnant women (29/425, 6.8% vs 5/510, 0.9%, P < 0.001). Of the geographic regions, Preah Sihanouk province (Southwest) had the highest anti-HCV prevalence among PLWHA (12.0%, P = 0.031). There was no significant geographic difference in anti-HCV prevalence among pregnant women. In multivariable analyses (data subset to PLWHA), HCV infection was significantly associated with having a family member positive for HCV (OR = 7.6 [95% CI: 1.01-57.84], P = 0.048) and a history of intravenous medication injection in the last 5 years (OR = 7.1 [95% CI: 2.79-18.10], P < 0.001). Conclusions: HCV infection is relatively common among Cambodian PLWHA, likely related to intravenous medication injection and intra-familial viral transmission. Systematic HCV testing and care among PLWHA (and possibly their family members) might be necessary. Setting up a surveillance system for HCV might also be beneficial for some geographical regions and populations.