The stem-loop III (SLIII) structure within the 5' untranslated region has been shown to be critical for internal initiation of translation of Hepatitis C virus (HCV). Using 'Single Strand Conformation Polymorphism (SSCP)' of the SLIII region we have investigated for natural mutations and demonstrated presence of some non-covariant changes in certain sub-domains. However, overall SLIII-RNA structure was found to be phylogenetically conserved. Additionally, by SSCP analysis we have determined the genotype of 50 HCV isolates collected from Southern India, 25 random samples were confirmed by DNA sequencing. Results showed the prevalence of genotype 1 in this part of India.