Hepatitis C virus (HCV) exhibits genotype-specific variations in geographical distribution as a consequence of drug and immune induced evolution. Present study was aimed at discerning the distribution and prevalence of the various genotypes and subtypes of HCV in southern India. The HCV positive patient's serum was collected from different hospitals and blood banks from the states of Kerala, Tamil Nadu, Andhra Pradesh and Karnataka. Among 114 HCV positive samples, we could find only 44 isolates that are found both positive in ELISA and RT-PCR. From these samples 5' untranslated region (5'UTR) were amplified, sequenced and sub typed. Analysis of 5'UTR region of the 44 isolates shows that, genotypes 1, 3, 4 and 6 are present with genotype 3 being the most frequent. The present study shows that HCV genotype 3 subtype B was the most prevalent, forming 47.7 % among the population in southern India. The present study urges for discovering novel therapeutic agents that should be specific to genotype 3 subtype B, for the management of HCV in southern India.