Human isolates of Mycobacterium collected in 16 different states of Brazil were submitted to PCR-restriction analysis (PRA) of a 439-bp fragment of the hsp65 gene with HaeIII and BstEII. Fourteen allelic variants not described in clinical isolates so far were observed among 36 (10%) of 356 Brazilian strains, including a new pattern for Mycobacterium scrofulaceum, M. intracellulare, and M. flavescens, two new patterns for M. fortuitum, three new patterns each for M. gordonae and M. terrae, and one new pattern for M. avium complex-like strains. Two unidentified strains each also presented a new pattern, strongly suggesting that Mycobacterium genotypes are distributed biogeographically. The PRA procedure was also performed with 43 reference isolates belonging to 34 species, adding a further six new patterns to the identification algorithm. A database containing the normalized restriction patterns of both enzymes was constructed. Patterns available on the Internet can be introduced into this database, which will make possible the comparison of genotypes from isolates from different parts of the world.