The mutation in KatG315 is found in the majority of isoniazid resistant strains worldwide, especially in areas with a high incidence of tuberculosis. A total of 138 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis consisting of 108 MDR (multidrug resistant) and 30 XDR (extensively drug resistant) isolated from patients in different regions of Belarus from 2007 to 2008 were screened by a PCR restriction fragment length polymorphism (RFLP) assay and sequencing. As a result, 97.8% prevalence of the KatG315 mutation was detected in all isolates from patients either actually or previously treated with tuberculosis. This mutation was not found in any of 9 INH-susceptible isolates and 2 standard strains of H37Rv and Academia included in the study. All isolates that contained the mutation in KatG315 were classified as MDR and XDR by a culture-based susceptibility testing method. Among the 30 XDR isolates, 15 (50%), 12 (40%), and 3 (10%) were classified into principal genetic groups (PGG) 1, 2, and 3, respectively. It is concluded that INH-resistant MTB were associated with the mutated KatG315 phenotype. The simplicity of the assay, with 100% specificity, permits its implementation in routine practice at clinical microbiology laboratories for first and fast screening of cultures. This method has potential application for rapid diagnosis of INH resistance due to KatG315 mutation.