Daclatasvir (DCV; BMS-790052) is a hepatitis C virus (HCV) NS5A replication complex inhibitor (RCI) with picomolar to low nanomolar potency and broad genotypic coverage in vitro. Viral RNA declines have been observed in the clinic for both alpha interferon-ribavirin (IFN-α-RBV) and IFN-RBV-free regimens that include DCV. Follow-up specimens (up to 6 months) from selected subjects treated with DCV in 14-day monotherapy studies were analyzed for genotype and phenotype. Variants were detected by clonal sequencing in specimens from baseline and were readily detected by population sequencing following viral RNA breakthrough and posttreatment. The major amino acid substitutions generating resistance in vivo were at residues M28, Q30, L31, and Y93 for genotype 1a (GT-1a) and L31 and Y93 for GT-1b, similar to the resistance substitutions observed with the in vitro replicon system. The primary difference in the resistance patterns observed in vitro and in vivo was the increased complexity of linked variant combinations observed in clinical specimens. Changes in the percentage of individual variants were observed during follow-up; however, the overall percentage of variants in the total population persisted up to 6 months. Our results suggest that during the 14-day monotherapy, most wild-type virus was eradicated by DCV. After the end of DCV treatment, viral fitness, rather than DCV resistance, probably determines which viral variants emerge as dominant in populations.