beta-lactamases from 259 strains of rapidly growing mycobacteria that included the third biovariant complex of Mycobacterium fortuitum, M. peregrinum, M. abscessus, M. chelonae, the M. chelonae-like organisms (MCLO), and M. smegmatis were analyzed by isoelectric focusing (IEF). All isolates produced acidic beta-lactamases with major band isoelectric points (pIs) between 4.4 and 6.0. Each of the 6 taxonomic groups exhibited 1 or 2 characteristic beta-lactamase IEF patterns. Heterogeneity among IEF patterns was evident in 5 of the 6 groups, however, and was greatest among the third biovariant complex of M. fortuitum. beta-lactamase patterns correlated with previously identified taxonomic subgroups of M. smegmatis and the third biovariant complex of M. fortuitum. beta-lactamase IEF analysis of MCLO strains isolated from two outbreaks demonstrated its possible usefulness for epidemiologic evaluation.