The objective was to determine the incidence, clinical significance, and epidemiology of the isolates of nonpigmented, rapidly growing mycobacteria (NPRGM) in Madrid, Spain. Patients with new isolates of NPRGM during 2005 were selected prospectively for review of clinical charts. Clinical significance was analyzed according internationally accepted criteria. Randomly amplified polymorphic DNA (RAPD) was used for the genotyping of the isolates. NPRGM were identified in 70 patients (1.51 cases/100,000 inhabitants). The species were M. abscessus (in 5 patients), M. chelonae (in 9), M. fortuitum (in 40), M. peregrinum (in 9), M. mageritense (in 5), M. mucogenicum (in 2), and M. alvei (in 1 patient). The isolates were clinically significant in 17 cases (24.3%, 0.39 cases/100,000 inhabitants): in 4 cases of M. abscessus, in 5 of M. chelonae, and in 9 of M. fortuitum. Only 10.7% of the respiratory isolates were significant, whereas 75% of the nonrespiratory ones were significant (p < 0.001). RAPD analysis showed no relationship among the 74 strains available for the study. No characteristic resistance pattern could be found, although 4 strains appeared to be resistant to amikacin. Significant isolates were mainly nonrespiratory ones. The most significant species was M. abscessus. No relationship between the various isolates was detected, ruling out interhuman transmission between these cases.