Sustained virological responses (SVR) by daclatasvir (DCV) and asunaprevir (ASV) therapy for genotype 1b hepatitis C virus (HCV) infected patients has been significantly affected by pre-existence of Y93 H resistance-associated variants (RAVs) in the non-structural protein 5A (NS5A) region. The aim of this study was to elucidate the dominancy of naturally occurring RAVs in viral quasispecies on treatment outcomes in patients with HCV. In total, 138 patients were prospectively selected from 152 patients treated with DCV and ASV, where evaluation of treatment outcomes at 12 weeks post-treatment was possible. Pre-treatment RAVs in the non-structural protein 3 and NS5A regions were detected by polymerase chain reaction (PCR)-Invader assays, and the ratio of Y93H RAVs in viral quasispecies was measured by quantitative PCR-Invader assay. Among 25 patients detected the Y93H RAV, the Y93H ratio was 1-25% in 5 patients, 26-75% in 7 patients, and ≥76% in 13 patients. Overall, SVR at 12 weeks after the completion of treatment (SVR12) was 91% (125/138), and those with Y93H ratios of <1%, 1-25%, 26-75%, and ≥76% were 99%, 100%, 71%, and 23%, respectively. Thus, the SVR12 decreased as the HCV Y93H ratio increased (P < 0.0001). The dominancy of pre-treatment RAVs of DCV and ASV affected its treatment outcomes, suggesting that evaluating the dominancy of HCV RAVs could be required for every other direct-acting antiviral agent treatments. J. Med. Virol. 89:99-105, 2017. © 2016 Wiley Periodicals, Inc.