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We compared a commercial line probe assay (INNO-LiPA™ HCV II, Innogenetics, N.V., Ghent, Belgium, distributed by Bayer Diagnostics) to an in-house 5′ untranslated region direct DNA sequencing method for genotyping hepatitis C virus (HCV). Initial evaluation demonstrated that the INNO-LiPA™ HCV II assay and sequencing assay assigned the same genotype for 110/132 (83.3%) patient specimens (98 subtype and 12 genotype only identifications). Following the initial evaluation, the INNO-LiPA™ HCV II assay was used routinely to genotype HCV from patient specimens submitted to our laboratory for genotyping (n = 1,739). During this second part of the study, novel line probe patterns have been noted and interpreted using the in-house direct sequencing assay. Reactivity at bands 1, 2, 3, 4, 5 and 8 (n = 4) or 1, 2, 3, 4, 6 and 7 (n = 2) represented HCV genotype 1. Reactivity at bands 1, 2, 5 and 9 (n = 1) represented HCV genotype 2. Reactivity at bands 1, 2, 5, 9 and 16 (n = 1) represented HCV genotype 4. Reactivity at bands 1, 2, 5, 9, 10, 11 (weak band) and 12 (n = 118) most likely represented HCV genotype 2b. This information should be of use to INNO-LiPA™ HCV II assay users. [Copyright &y& Elsevier]