The HCV subtype 3a is unique in producing the symptoms as it causes hepatic steatosis, which is not commonly observed in other genotypes of HCV. The molecular difference between subtypes 3a and 3b remains unexplored. This study tried to explore this difference between these two subtypes. The antagonist bound conformation of NS5B protein of both subtypes 3a and 3b of HCV were modeled using the crystal structure of ‘4aep’ (NS5B, subtype 2a) through homology modeling. Four HCV competitive antagonists [(1) IPC; (2) JPC; (3) 79Z; (4) 26F] were used to find the differences between the subtypes 3a and 3b by the use of docking. Although it showed 88% sequence similarity, however, these were observed to be substantially different, while analyzing the patterns of the intermolecular hydrogen... bonding, disulfide bonding, non-conserved residue interactions, individual charged residues interaction as well as conformational changes. About 5 interaction sites and two conserved hydrophilic pockets were found to be crucial for antagonist binding. Further, the invariant three Arg residues (Arg158, Arg386, and Arg394) were seen to differently influence the ligand binding, while the involvements of disulfide bonds of the two subtypes in the interactions with drugs were also different. Such information will be crucial for future development of selective subtype specific drugs against the HCV.