The dried blood spot (DBS) samples are a useful resource for viral DNA isolation and important in increasing access to HBV diagnosis. However, the choice of the DNA extraction method is crucial for reliable results. We compared the reliability of four DNA extraction methods using DBS samples for the qualitative and quantitative de tection of HBV. A panel of serially diluted HBV DNA in whole blood was spotted onto filter paper (Whatman 903 paper and Whatman FTA cards). Four methods were used to extract DNA: QIAamp® DNA Blood Mini Kit (Qiagen); High Pure Viral Nucleic Acid Kit (Roche); Invisorb Spin Blood Midi Kit (Invitek), and DBS Genomic DNA Isolation Kit (Norgen Biotek). Two qualitative PCRs for the core and surface gene regions of HBV were used, and in-house real-time PCR was also evaluated. It was possible to detect HBV DNA using all extraction and PCR protocols. The lowest limit of detection was found using Whatman 903 paper, Roche extraction, and qualitative PCR (20 copies of HBV DNA per ml) for the surface/polymerase HBV region. In the case of in-house real-time PCR, the lowest limit of detection was found using both Roche and Qiagen assays (estimated 2 × 103 copies per ml). These results suggest the importance of both the extraction method and PCR protocol in detecting HBV DNA in DBS. This study provides insights into the utility of DBS samples in HBV molecular diagnosis and their feasibility in low resource areas where cold storage and transportation may be difficult.