We have previously demonstrated homology between a 181-bp fragment of IS6110 and DNA from mycobacteria other than tuberculosis-causing mycobacteria (MOTT). Genomic DNA from 14 strains of MOTT was digested with PvuII and was hybridized with a probe derived from the 181-bp fragment and the INS1/INS2 international standard probe at high stringency. Multiple banding patterns were obtained from isolates of M. avium-M. intracellulare, M. fortuitum, M. kansasii, and M. malmoense. Differences in the banding patterns between and within species were obtained. This suggests that mycobacteria possess a family of IS3-like elements. The species of isolates suspected of being M. tuberculosis must be carefully determined before IS6110 restriction fragment length polymorphism analysis, and caution must be used in designing and evaluating diagnostic PCR tests based on this element.