Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. Polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of the gene enconding for the 65 kDa heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype M. kansasii isolates. In the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at Mycobacteria Laboratory of the Instituto Adolfo Lutz in the period 1995-1998. A total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as M. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. Of the 296 M. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by PRA-hsp65. Hundred eight two (98.9%) were classified as M. kansasii type I. Two isolates were classified as type II and III and five isolates were characterized as other Mycobacterium species. Clinical isolates of M. kansasii in the state of Sao Paulo was almost exclusively subtype I regardless of HIV status.