The prevalence of mutations at amino acid (aa) position 315 in the katG gene of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in The Netherlands and the mutation's association with the level of INH resistance, multidrug resistance, and transmission were determined. Of 4288 M. tuberculosis isolates with available laboratory results, 295 (7%) exhibited INH resistance. Of 148 aa 315 mutants, 89% had MICs of 5–10 µ g/mL, whereas 75% of the other 130 INH-resistant strains had MICs of 0.5–1 µ g/mL. Of the aa 315 mutants, 33% exhibited monodrug resistance, compared with 69% of other INH-resistant strains ( P <.0001). Multidrug resistance was found among 14% of the aa 315 mutants and 7% of the other INH-resistant strains ( P > .05). The probability of being in an IS 6110 DNA restriction fragment length polymorphism cluster was similar for aa 315 mutants and INH-susceptible strains, but the probability was reduced in other INH-resistant strains. Thus, aa 315 mutants lead to secondary cases of tuberculosis as often as INH-susceptible strains do.