Introduction Data on hepatitis B virus (HBV) are limited in developing countries. The study goal was to evaluate the prevalence and factors associated with HBV infection in Peru, highlighting its relationship with consistent condom use. Methods Data from two different surveys performed in 28 mid-sized Peruvian cities were used. Participants aged 18–29 years were selected using a multistage cluster sampling. Information was collected through a validated two-part questionnaire. The first part (face-to-face) concerned demographic data, while the second part (self-administered using handheld computers) concerned sexual behaviour. Hepatitis B core antibody (anti-HBc) was tested in 7000 blood samples. Prevalences and associations were adjusted for sample strata, primary sampling units and population weights. Results: Anti-HBc prevalence was 5.0% (95% CI 4.1% to 5.9%), with the highest prevalence among jungle cities: 16.3% (95% CI 13.8% to 19.1%). Anti-HBc positivity was associated with geographic region (highlands OR=2.05; 95% CI 1.28 to 3.27, and jungle OR=4.86; 95% CI 3.05 to 7.74; compared to coastal region); and age at sexual debut (OR=0.90; 95% CI 0.85 to 0.97). Consistent condom use was associated with lower prevalence (OR=0.34; 95% CI 0.15 to 0.79) after adjusting for sex, geographic region, education level, lifetime number of sex partners, and age at sexual debut. Conclusion: Residence in highlands or jungle cities is associated with higher anti-HBc prevalences, whereas increasing age at sexual debut was associated with lower prevalences. Consistent condom use was associated with decreased risk of anti-HBc. These findings emphasise the need of vaccination especially in jungle population, and imply that condom use promotion might be a strategy to prevent HBV infection.