To develop a strategy for rapid species assignment and strain differentiation of Mycobacterium avium complex (MAC) organisms, the sequence of a 360-bp region of the gene (hsp65) encoding a 65-kDa heat shock protein was determined for 56 isolates, including 21 patient isolates and 35 reference strains. Eleven hsp65 alleles were identified, and there was no sharing of alleles between strains classified as M. avium and Mycobacterium intracellulare based on serovar and species-specific DNA hybridization probes. Phylogenetic analysis showed that 30 strains had one of two hsp65 alleles which were found in known M. avium organisms, 23 strains had one of six alleles allied with known M. intracellulare organisms, and three MAC isolates had one of three hsp65 alleles that differed substantially from the consensus M. avium and M. intracellulare hsp65 sequences. Estimates of strain relationships based on the sequences of hsp65 and the 16S-23S ribosomal DNA internal transcribed spacer were similar. Automated DNA sequencing of a 360-bp region of the hsp65 gene from MAC organisms provides a rapid and unambiguous marker system for strain differentiation and permits specific assignment of these acid-fast organisms for diagnostic purposes.