Objective: To assess the seroprevalence of hepatitis B virus (HBV), syphilis, and HIV and associated risk factors in pregnant women and their male partners from six indigenous populations of the Peruvian Amazon Basin. Methods: A cross-sectional study was performed in six indigenous populations from the Peruvian Amazon Basin. Blood samples were obtained and tested for HBV (antibodies to the hepatitis B core antigen (anti-HBc) and hepatitis B surface antigen (HBsAg)), for syphilis (rapid plasma reagin and microhemagglutination assay for Treponema pallidum antibodies), and for HIV (ELISA and indirect immunofluorescence test). A survey was also performed to identify associated risk factors. Results: One thousand two hundred and fifty-one pregnant women and 778 male partners were enrolled in the study. The seroprevalence of anti-HBc in pregnant women was 42.06% (95% confidence interval (CI) 39.28-44.85%) and in their male partners was 54.09% (95% CI 50.32-57.86%). The seroprevalence of HBsAg in pregnant women was 2.11% (95% CI 0.78-3.44%) and in their male partners was 3.98% (95% CI 1.87-6.08%). The seroprevalence of syphilis in pregnant women was 1.60% (95% CI 0.86-2.33%) and in their male partners was 2.44% (95% CI 1.22-3.66%). HIV seroprevalence in pregnant women was 0.16% (95% CI 0.02-0.58%) and in their male partners was 0.29% (95% CI 0.04-1.03%). Sexual risk factors were strongly related to blood markers of syphilis and HBV. Conclusions: Hepatitis B was found to be hyperendemic and strongly related to sexual factors, suggesting an important sexual component in the transmission of the disease in the populations studied. Syphilis was found to have an endemicity in pregnant women above the national level and this may be indicative of high mother-to-child transmission. HIV has started to show its presence in indigenous populations of the Amazon Basin and the results suggest the epidemic is concentrated. © 2012 International Society for Infectious Diseases.