A previous limited study demonstrated that Mycobacterium tuberculosis isolates with a mutation at amino-acid position 315 of katG (Δ315) exhibited high-level resistance to isoniazid and were more frequently resistant to streptomycin. In the present study, isoniazid-resistant M. tuberculosis isolates from 8332 patients in The Netherlands (1993–2002) were screened for the Δ315 mutation. Isoniazid resistance was found in 592 (7%) isolates, of which 323 (55%) carried Δ315. IS6110 restriction fragment length polymorphism analysis showed that Δ315 isolates occurred in clusters, suggesting recent transmission, at the same frequency as isoniazid-susceptible isolates. In contrast, other isoniazid-resistant isolates clustered significantly less frequently. Δ315 isolates were high-level isoniazid-resistant, streptomycin-resistant and multidrug-resistant significantly more often, and may have a greater impact on public health, than other isoniazid-resistant isolates.