Abstract Background The 9.6 kb long RNA genome of Hepatitis C virus (HCV) is under the control of RNA dependent RNA polymerase, an error-prone enzyme, for its transcription and replication. A high rate of mutation has been found to be associated with RNA viruses like HCV. Based on genetic variability, HCV has been classified into 6 different major genotypes and 11 different subtypes. However this classification system does not provide significant information about the origin of the virus, primarily due to high mutation rate at nucleotide level. HCV genome codes for a single polyprotein of about 3011 amino acids which is processed into structural and non-structural proteins inside host cell by viral and cellular proteases. Results We have identified a conserved NS4A protein sequence for HCV genotype 3a reported from four different continents of the world i.e. Europe, America, Australia and Asia. We investigated 346 sequences and compared amino acid composition of NS4A protein of different HCV genotypes through Multiple Sequence Alignment and observed amino acid substitutions C₂₂, V₂₉, V₃₀, V₃₈, Q₄₆ and Q₄₇ in NS4A protein of genotype 1b. Furthermore, we observed C₂₂ and V₃₀ as more consistent members of NS4A protein of genotype 1a. Similarly Q₄₆ and Q₄₇ in genotype 5, V₂₉, V₃₀, Q₄₆ and Q₄₇ in genotype 4, C₂₂, Q₄₆ and Q₄₇ in genotype 6, C₂₂, V₃₈, Q₄₆ and Q₄₇ in genotype 3 and C₂₂ in genotype 2 as more consistent members of NS4A protein of these genotypes. So the different amino acids that were introduced as substitutions in NS4A protein of genotype 1 subtype 1b have been retained as consistent members of the NS4A protein of other known genotypes. Conclusion These observations indicate that NS4A protein of different HCV genotypes originally evolved from NS4A protein of genotype 1 subtype 1b, which in turn indicate that HCV genotype 1 subtype 1b established itself earlier in human population and all other known genotypes evolved later as a result of mutations in HCV genotype 1b. These results were further confirmed through phylogenetic analysis by constructing phylogenetic tree using NS4A protein as a phylogenetic marker.