Nocardia identification required laborious and time-consuming phenotypic and chemotaxonomic methods until molecular methods were developed in the mid-1990s. Here we reassessed the capacity of PCR-restriction enzyme pattern analysis (PRA) of the hsp65 gene to differentiate Nocardia species, including 36 new species. Our results confirm that hsp65 PRA must no longer be used for Nocardia species identification, as many species have the same restriction pattern. We then compared sequencing-based strategies using an hsp65 database and a 16S rRNA database and found that the hsp65 region contained sufficient polymorphisms for comprehensive Nocardia species identification.