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Molecular epidemiology of Mycobacterium tuberculosis is useful for understanding disease transmission dynamics, and to establish strategic measures for TB control and prevention. The aim of this study was to analyze clinical, epidemiological and molecular characteristics of MTBC clinical isolates from Santa Catarina state, southern Brazil. During one-year period, 406 clinical isolates of MTBC were collected from Central Laboratory of Public Health and typed by spoligotyping. Demographic and clinical data were collected from the Brazilian National Mandatory Disease Reporting System. The majority of cases occurred in highest population densities regions and about 50% had some condition associated with TB. Among all isolates, 5.7% were MDR, which showed association with drug addiction. LAM was the most predominant lineage with 47.5%, followed by the T superfamily with 25.9% and Haarlem with 12.3%. The MST showed two major groups: the first was formed mainly by the LAM lineage and the second was mainly formed by the T and Haarlem lineages. Others lineages were distributed in peripheral positions. This study provides the first insight into the population structure of M. tuberculosis in SC State. Spoligotyping and other genotyping analyses are important to establish strategic measures for TB control and prevention.