The prevalence of naturally occurring mutations in hepatitis C virus associated with resistance to protease inhibitors in chronically infected patients has not been reported in Brazil. The NS3 serine protease domain was sequenced in 114 therapy-naïve patients infected with subtype 1a (n = 48), 1b (n = 53), or 3a (n = 13). A V36L mutation was observed in 5.6% patients infected with subtype 1b and in all isolates of the 3a subtype, and a T54S mutation was detected in 4.1% of isolates of subtype 1a. In conclusion, the presence of variants carrying mutations associated with resistance to protease inhibitors in therapy-naïve patients may be important for future therapeutic strategies.