The incidence of tuberculosis (TB) is increasing in high-risk populations in Uruguay, possibly owing to emerging resistance. Mycobacterial interspersed repetitive units (MIRU) genotyping and katG sequence analysis of isoniazid (INH) resistance-associated mutations were performed in 45 INH-resistant Mycobacterium tuberculosis isolates in Uruguayan patients. The genotype distribution among INH-resistant isolates shares features of that of neighbouring countries, with a predominance of Latin American and Mediterranean, T and Haarlem genotypes, although the S genotype was particularly frequent among our isolates. Forty-four per cent of INH-resistant strains harboured the S315T mutation in katG; we found novel katG mutations (W321X, G269T, P232R and G221Wfs1) that could explain INH resistance.