This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.