The study of hepatitis C virus (HCV) genotypes/subtypes, quasispecies and recombinants obtained by virus genome sequencing are important for epidemiological studies, to trace the source of infection, for development of new direct acting antivirals (DAAs) therapy and for understanding antiviral selection pressures. The HCV NS5B gene encodes a polymerase, which is responsible for virus replication and is a potential target for the development of antiviral agents. Many studies for classification of HCV use a particular segment of the NS5B gene, in addition to other specific regions, and phylogenetic analysis. Actually, some nucleoside/nucleotide analogues and non-nucleoside inhibitors target NS5B protein. This review focuses on HCV variability, phylogenetic analysis and the role of NS5B in the virus-host interactions.