Hepatitis C virus (HCV) quasispecies heterogeneity was assessed in pretreatment sera of 22 patients treated with interferon (IFN). Quasispecies heterogeneity was quantitated by analysis of 490 hypervariable region 1 (HVR1) clones using gel shift analysis (GSA), which allowed determination of two components of HCV quasispecies heterogeneity: genetic complexity (number of variants) and genetic diversity (mean genetic distance between variants). HCV genotype and pretreatment RNA titer were similar between responders (n = 12) and nonresponders (n = 10). GSA correlated well with nucleotide sequencing for estimating HCV genetic diversity (R = .952; P < .001). A positive correlation of .666 (P < .001) was observed between genetic diversity and complexity, suggesting that these components of quasispecies heterogeneity were related. However, while there were no significant differences in pretreatment complexity or genetic diversity among response groups, higher pretreatment genetic diversity was predictive of response failure (P = .041). Assessment of HVR1 heterogeneity by GSA revealed important relationships between quasispecies genetic diversity, complexity, and response to IFN therapy and suggested a need for analysis of other HCV genes.