To validate a commercially available line probe assay for samples containing the infrequently found hepatitis C virus (HCV) genotype 5a, we sequenced a 511-nucleotide fragment of the NS4b region of Belgian and South African HCV genotype 5a samples. Phylogenetic analysis of the sequence data was performed. For the 77 HCV genotype 5a samples collected, there was 100% concordance between the genotype assignment by the line probe assay and the genotyping based on nucleotide sequencing, despite sequence heterogeneity in the probe binding sites of some samples.