Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5' untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n=312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n=190), 12.8% type 2 (n=40), 20.2% type 3 (n=63), 0.3% type 4 (n=1), and 0.3% other (n=1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections.