Summary A major threat to tuberculosis (TB) control programs is the emergence of drug resistant Mycobacterium tuberculosis strains that cause TB that cannot be cured by standard anti-TB drug regimens. Because few data exist on MDR-TB in this region of the country, we performed an epidemiologic study that combined conventional and molecular analysis of MDR-TB cases from Rio Grande do Sul (RS) that were diagnosed in this period and included cases that were under treatment with second line drug schemes. Included were 121 MDR cases and sequencing of rpo B and kat G showed that 106 (87.6%) strains were mutated in rpo B and 97 (80.2%) in kat G. Spoligotyping demonstrated that the LAM genotype was predominant ( n  = 70, 57.8%) and included the largest group composed by 22 (18.1%) strains with the LAM5 ST93 genotype. Other main genotypes belonged to the families T ( n  = 22, 18.2%), U family ( n  = 16, 13.2%), Haarlem ( n  = 5, 4.1%) and X ( n  = 1, 0.8%). Genotyping by IS 6110- RFLP analysis showed 51 distinct fingerprints, 38 (31.4%) of these observed only once and the other 13 patterns being shared among the rest of the isolates ( n  = 83, 68.6%). Among the 22 strains that were LAM5 ST93, only two had different IS 6110 -RFLP genotypes. In conclusion, there exists a high degree of M. Tuberculosis genotype clustering among MDR-TB cases in Rio Grande do Sul. Moreover, we observed a large MDR-TB outbreak.