Background: The aim of this study was to investigate the significance of mutation in codon 315 of katG gene and its correlation with high-level of resistance to isoniazid, nuclotide and amino acid changes in mycobacterium tuberculosis (MTB) isolates randomly collected from sputums of 42 patients with active pulmonary tuberculosis in different regions of Belarus. Materials and Methods: Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA Extraction, katG gene amplification, and DNA sequencing analysis were performed. Results: Six isolates (14%) bearing multi-mutations in three codons (309,315 and 316), 26 Isolates (61.9%) demonstrated multi-mutations in all or two of the above codons, and 8 (19%) were found to have a single mutation in 315. Four types of mutations were identified in codons 315: AGC→ACC (n=36)85%, AGC→AGG (n=1) 2.3%, AGC→AAC (n=2) 4.7%, AGC→GGC (n=1) 2.3%, one type of mutation in 316: GGC→AGC (n=18)41.4%, and four types of mutations in 309: GGT→GGT (n=7)16.1%, GGT→GCT (n=4)9.2%, GGT→GTC (n=3)6.9%, GGT→GGG (n=1)2.7%. In 2 (4.7%) isolates mutations were identified in codons 463, 357, and in codons 454, 357 respectively. Conclusion: MTB in patients from Belarus were found to have high-level resistance to isoniazid in the isolates with mutations in codon 315 (> 10 µg/mL). (Tanaffos 2007; 6(3): 11-19)