A total of 129 reference and clinical strains of rapidly growing mycobacteria (RGM) belonging to 10 taxonomic groups were studied for restriction fragment length polymorphism patterns from a PCR-amplified 439-bp segment of the 65-kDa heat shock protein (HSP) gene. Of 24 endonucleases evaluated, restriction fragment length polymorphism patterns produced by HaeIII and BstEII and then by AciI and CfoI gave the best separation. Sixty percent of all RGM taxa studied were differentiated by HaeIII digests alone. Single unique patterns were observed with HaeIII and/or BstEII for Mycobacterium fortuitum (100%), M. chelonae (94%), M. abscessus (96%), M. smegmatis (100%), M. mucogenicum (formerly the M. chelonae-like organism) (100%), and the sorbitol-negative third biovariant of M. fortuitum (100%). Evidence is presented in support of two subgroups within M. peregrinum, M. smegmatis, and the unnamed third biovariant of M. fortuitum (sorbitol positive and sorbitol negative). PCR-based technology provides a rapid, accurate system for the identification of clinically important species of RGM which should be particularly useful for reference laboratories.