Background: The prevalence of HBV infection and HBV genotypes varies from country to country, and the role of HBV genotypes in the presence of HBV in the placenta and fetus has never been explored. This study was conducted to (1) identify HBV genotypes, and their frequencies, that infected Northern Thai pregnant women; (2) evaluate the association between HBV genotypes and the detection rate of HBV DNA in the placenta and fetus; (3) evaluate the association between specific mutations of the HBV genome and HBV DNA detection in placental tissue; and (4) identify the mutation of the HBV genome that might occur between maternal blood, placenta, and cord blood. Methods: Stored samples of the maternal blood, placental tissue, and cord blood that were collected from 145 HBsAg-positive pregnant Thai women were analyzed to identify HBV DNA. Results: Approximately 25% of infected mothers had fetal HBV DNA detection, including cases with concomitant HBV DNA detection in the placenta (77.3%). A total of 11.7% of cases with placental detection had no HBV DNA detection in the maternal blood, indicating that the placenta could be a site of HBV accumulation. Of the 31 HBV-positive blood samples detected by nested PCR, the detected strains were subgenotype C1 (77.4%), subgenotype B9 (9.7%), and subgenotype C2, B2, B4, and recombinant B4/C2 (3.2% for each). Genotype B had a trend in increased risk of placental HBV DNA detection compared to genotype C, with a relative risk of 1.40 (95% CI: 1.07-1.84). No specific point mutation had a significant effect on HBV DNA detection in placental tissue. Mutation of C454T tended to enhance HBV DNA detection in placental tissue, whereas T400A tended to have a lower detection rate. No mutation was detected in different sample types collected from the same cases. Conclusions: HBV DNA detection in the fetus was identified in approximately 25% of HBV-positive mothers, associated with the presence of HBV in the placenta in most cases. The placenta could possibly be a site of HBV accumulation. Subgenotype C1 was the most common subgenotype, followed by subgenotype B9. HBV genotype B possibly had a higher trend in intrauterine detection than HBV genotype C. Mutation is unlikely to occur during intrauterine exposure.