ABO and Rh blood groups play a vital role in blood transfusion safety and clinical practice and are thought to be linked with disease susceptibility. The results from previous studies that focused on the association between blood groups and HBV infection remain controversial. China has the world's largest burden of HBV infection. We assessed the distribution of ABO/Rh blood groups in Chinese adults and examined the association between these groups and HBV infection. We did a nationwide cross-sectional study using data from a physical check-up programme from 31 provinces examined between 2010 and 2012. ELISA was used to test for HBsAg in serologic samples. Multivariable logistic regression was used to estimate aOR of the association between ABO and Rh blood groups and HBV infection. Among 3 827 125 participants, the proportion of participants with blood group A was highest (30.54%), followed by O (30.37%), B (29.42%) and AB (9.66%). A total of 38 907 (1.02%) were Rh-D negative. The prevalence of HBsAg in blood groups O, A, B and AB were 6.34%, 5.55%, 5.18% and 5.06%, respectively. HBsAg prevalence was 5.65% in Rh-D-positive and 3.96% in Rh-D-negative participants. After controlling for other potential risk factors, multivariate models showed that participants with blood group O (adjusted OR = 1.22, 95% CI: 1.20-1.25) were at higher risk of HBV infection compared with group AB. Rh-D-positive participants (adjusted OR = 1.44, 95% CI: 1.37-1.52) were at higher risk of HBV infection than Rh-D-negative participants. The associations between ABO/Rh blood groups and HBV infection were similar in subgroup analysis. The proportions of O, A, B and AB blood groups were approximately 3:3:3:1, and nearly 1 in 100 people was Rh-D negative among Chinese adults. Blood group O and Rh-D positivity were both associated with increased HBV infection. The risk of HBV infection and blood safety should be taken into consideration in clinical practice, especially when transfusing those with blood group O. Awareness and prevention of HBV infection is of particular importance for individuals with blood group O.