In addition to its known status as a disseminated disease in HIV-positive patients, Mycobacterium avium complex (MAC) is increasingly recognized as a causative pathogen of respiratory disease in HIV-negative patients. MAC is divided into Mycobacterium avium, and the less-epidemiologically studied Mycobacterium intracellulare. Genetic typing for M. intracellulare using variable number of tandem repeats (VNTR) has not yet been developed. The aim of this study was to identify VNTR loci in the genome of M. intracellulare and apply them as an epidemiological tool to clinical isolates. Here, we identified 25 VNTR loci on the M. intracellulare genome, of which 16 showed variations among clinical isolates in the number of tandem repeat motifs. Among the 74 M. intracellulare isolates, 50 genotypes were distinguished using the 16 VNTR loci, resulting in a Hunter Gaston's discriminatory index of 0.988. Moreover, all 16 VNTR loci were stable in different sets of isolates recovered within time intervals ranging from 2 to 1551 days from 14 separate patients. These results indicate that for use as epidemiological markers of M. intracellulare, the loci in this VNTR assay are highly discriminating and stable over time.