Non-nucleoside inhibitors of the RNA-dependent RNA polymerase of the hepatitis C virus that are based on a benzimidazole or indole scaffold have been reported to interact with thumb domain 1 of the enzyme. Escape mutants that confer in vitro resistance to these inhibitors map to amino acids P495, P496 or V499. We here report a novel resistance mutation (T389S/A) that was identified following resistance selection with the benzimidazole non-nucleoside polymerase inhibitor JT-16 in HCV Con-1 subgenomic replicon (genotype 1b). This JT-16 resistant replicon retained wild-type susceptibility to protease inhibitors and nucleoside polymerase inhibitors. Replicons that carry mutations T389A and T389S have moderate levels of resistance to JT-16 (7- and 13-fold, respectively). Mutation P495A is associated with high-level (44-fold) resistance. Surprisingly, this previously reported ‘key’ mutation for benzimidazole resistance, P495A, was detected in only 15% of the resistant population. Furthermore, the replication fitness of the T389S mutant was significantly higher than that of the P495A mutant. By means of molecular modeling a structural hypothesis was formulated to explain the emergence of the T389S/A mutation in the JT-16 resistant replicon. Our data demonstrate that low-level resistant, but fit, variants can develop during in vitro resistance selection with the benzimidazole inhibitor JT-16. Moreover, different substitutions to the benzimidazole scaffold can affect the (pattern of) resistance mutations that emerge during resistance selection.