A considerable number of patients infected with Hepatitis C virus subtype 1b (HCV‐1b) do not respond to pegylated interferon/ribavirin combination therapy. In this study we explored a useful factor(s) to predict treatment outcome. A total of 47 HCV‐1b‐infected patients were treated with pegylated interferon/ribavirin for 48 weeks. Sera of the patients were examined for the entire NS5A sequence of the HCV genome, HCV RNA titers and anti‐NS5A antibodies. According to their responses, the patients were divided into two groups, early viral responders who cleared the virus by week 16 (EVR[16w]) and those who did not (Non‐EVR[16w]). The mean number of mutations in the V3 region (aa 2356 to 2379) or that in the V3 region plus its N‐terminally flanking region, which we refer to as interferon/ribavirin resistance‐determining region (IRRDR; aa 2334 to 2379), of NS5A obtained from the pretreatment sera was significantly larger for EVR(16w) compared with Non‐EVR(16w). Moreover, HCV‐1b isolates with ≥5 mutations in V3 or those with ≥6 mutations in IRRDR were almost exclusively found in EVR(16w). Also, the presence of detectable levels of anti‐NS5A antibodies in the pretreatment sera was closely associated with EVR(16w). In conclusion, a high degree of sequence variation in V3 (≥5) or IRRDR (≥6) and the presence of detectable levels of anti‐NS5A antibodies in the pretreatment sera would be useful factors to predict EVR(16w). On the other hand, a less diverse sequence in V3 (≤4) or IRRDR (≤5) together with the absence of detectable anti‐NS5A antibodies could be a predictive factor for Non‐EVR(16w).