Genotyping of hepatitis B virus (HBV) is important for tracking HBV infections, prognosticating the development of severe liver disease, and predicting outcomes of therapy. Current genotyping methods can be laborious and costly and rely on subjective data interpretation. To identify less expensive but equally reliable alternatives, we compared "gold standard" sequencing to a novel mass spectrometry approach. Sera from individuals with acute or chronic HBV infection (n = 756), representing all genotypes, were used to PCR amplify the HBV S gene. All amplicons were subjected to base-specific cleavage and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The resulting mass peak patterns were used to identify HBV genotype by automated comparison to peak patterns simulated from reference sets of HBV sequences of known genotypes. The MALDI-TOF MS data and phylogenetic analysis of HBV sequences produced completely concordant results. Several parameters such as genetic relatedness of tested HBV variants to the reference set, chronic infections, and the quality of PCR products can lower the MS score but never affected the accuracy of the genotype call. This new streamlined MS-based method provides for rapid and accurate HBV genotyping, produces automated data reports, and is therefore suitable for routine use in diagnostic settings.