Genetic heterogeneity is a hallmark of the hepatitis C virus, as a result largely of the infidelity of viral RNA–dependent RNA polymerase. Random nucleotide substitutions are introduced at a very high rate. The existence of genotypes was confirmed by statistical and mathematical techniques, and the relation of the genotypes to each other has been determined. There are six major genotypes, each with multiple subtypes. Isolates of the same genotype have an average sequence homology of 95%, but different genotypes have sequence similarity of approximately 65% on average. The nucleotide sequence in portions of the hepatitis C viral genome, including the 5′ noncoding region, part of the core gene, and other nonstructural proteins, is highly conserved. Genotype analysis typically utilizes these highly conserved regions. There are many techniques for determining viral genotype, and in general, concordance between techniques is good. Methods most commonly used for assigning hepatitis C virus (HCV) genotypes in clinical practice include restriction fragment length polymorphism analysis and the reverse hybridization line probe assay (LiPA; Innogenetics, Ghent, Belgium). The worldwide distribution of HCV genotypes has been determined; some genotypes are highly characteristic of certain areas. The most common subtypes, 1 and 2, are less genetically diverse than the others and are more widely distributed. The impact of genotype on disease course is controversial, but recent data suggest that there is a genotype-dependent differential response to therapy. Quasispecies refers to evolution of a highly related but genetically heterogeneous population of HCV isolates. The pathobiological and clinical implications of HCV quasispecies are poorly understood.