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Prevalence of Hepatitis B is relatively low in developed European countries. However specific subpopulations may exist within each country with markedly different Hepatitis B burden. Roma minority is very numerous in Slovakia and their lifestyle is completely different to non-Roma population. The aim of this study is to map Hepatitis B prevalence in Roma and compare it to non-Roma population and to explore potential socio-economic and health related risk factors. Cross-sectional epidemiology study was performed in Slovakia that included randomly sampled Roma population and geographically corresponding random sampled non-Roma population. Comprehensive questionnaire about risk factors was administered and blood samples were drawn for Hepatitis B serology and virology tests. Altogether 855 participants were included. Global Hepatitis B surface Antigen (HBsAg) positivity rate was 7.7% (i.e., active Hepatitis B) and anti Hepatitis B core IgG antibody (antiHBcIgG) positivity rate was 34.6%. Roma population had significantly higher prevalence of Hepatitis B, both active chronic infection (12.4%; 95% Confidence Interval (CI) 9.58%⁻15.97% versus 2.8%; 95% CI 1.56%⁻4.91%; p < 0.0001) and antiHBcIgG positivity (52.8%; 95% CI 48.17%⁻57.44% versus 25.9%; 95% CI 12.56%⁻20.02%; p < 0.0001) Main risk factors for HBsAg positivity were Roma ethnicity, male sex and tattoo., Conclusion: There is a very high prevalence of Hepatitis B in Roma communities in Slovakia, with potential for grave medical consequences.