To investigate whether single nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) 3 and 9 affect the susceptibility of hepatitis B virus (HBV) intrauterine transmission, we genotyped 399 neonates for TLR3 (c.1377C/T) [rs3775290] and TLR9 (G2848A) [rs352140] using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). A femoral venous blood sample was obtained from these subjects. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were measured using chemiluminescence immunoassay kits and hepatitis B virus DNA (HBV DNA) levels were determined by fluorescence quantitative PCR assay. Our results showed that when adjusting for maternal HBeAg, maternal HBV DNA and mode of delivery, allele ‘T’ for SNP c.1377C/T was significantly associated with HBV intrauterine transmission susceptibility [adjusted OR (aOR) 0·55, 95% confidence interval (CI) 0·34–0·91, P = 0·020] and the TT genotype decreased the risk of HBV intrauterine transmission (aOR 0·28, 95% CI 0·09–0·91, P = 0·033). Allele ‘A’ for SNP G2848A was significantly associated with HBV intrauterine transmission susceptibility (aOR 0·62, 95% CI 0·39–1·00, P = 0·048) and the GA genotype protected neonates from HBV intrauterine transmission (aOR 0·45, 95% CI 0·22–0·93, P = 0·031). The TLR3 (c.1377C/T) and TLR9 (G2848A) polymorphisms may be relevant for HBV intrauterine transmission susceptibility, although the reduction in risk to HBV intrauterine transmission is modest and the biological mechanism of the observed association merits further investigation.