The determination of HCV genotypes, subtypes and isolates has been helpful in understanding the evolution and the epidemiology of the virus, and is an important factor in the pre-treatment evaluation. A new simpler and automated sequencing based system has been developed recently, the Visible Genetics TruGene™ Hepatitis C Assay. The aim of the study was to compare this new genotyping assay with reverse hybridization based Innogenetics INNO-LiPA HCV II assay that is used most commonly. Eighty-eight HCV-RNA positive patients were enrolled and divided in four groups: 26 hemodialysed patients, 30 untreated patients with chronic HCV hepatitis, 12 IFN non-responder patients with chronic HCV hepatitis, 20 asymptomatic HCV positive subjects. The 5′-UTR region was amplified by RT-PCR and the nucleotide sequences determined by the TruGene™ assay. In parallel, the amplicons were also tested by INNO-LiPA. Concordant results were obtained in 80 out of 88 cases (90.9%). The new assay allowed to genotype 2 samples not typed by LiPA as 1b and 2a/c. The new system also allowed the subtyping of 3 untypable samples, classified as genotype 1 by INNO-LiPA, as genotype 1b (1 sample) and, as genotype 4 (2 samples). The difference between these genotype 4 isolates and the closest genotype 1 isolate was 6 nucleotides. One LiPA genotype 1a sample was typed as 1b and 2 genotype 1b samples were all typed as 1a by the sequence analysis. In conclusion, the new assay is a sensitive and rapid method that is suitable for accurate large-scale genotyping. J. Med. Virol. 63:17–21, 2001. © 2001 Wiley-Liss, Inc.