Hepatitis C viruses (HCVs) display a high level of sequence diversity and are currently divided into six genotypes. A line probe assay (LiPA), which targets the 5' untranslated region (5'UTR) of the HCV genome, is widely used for genotyping. However, this assay cannot distinguish many genotype 6 subtypes from genotype 1 due to high sequence similarity in the 5'UTR. We investigated the accuracy of a new generation LiPA (VERSANT HCV genotype 2.0 assay), in which genotyping is based on 5'UTR and core sequences, by testing 75 selected HCV RNA-positive sera from Southeast Asia (Vietnam and Thailand). For comparison, sera were tested on the 5'UTR based VERSANT HCV genotype assay and processed for sequence analysis of the 5'UTR-to-core and NS5b regions as well. Phylogenetic analysis of both regions revealed the presence of genotype 1, 2, 3, and 6 viruses. Using the new LiPA assay, genotypes 6c to 6l and 1a/b samples were more accurately genotyped than with the previous test only targeting the 5'UTR (96% versus 71%, respectively). These results indicate that the VERSANT HCV genotype 2.0 assay is able to discriminate genotypes 6c to 6l from genotype 1 and allows a more accurate identification of genotype 1a from 1b by using the genotype-specific core information.