RD(Rio) is a novel Mycobacterium tuberculosis lineage of the Latin American-Mediterranean (LAM) family. LAM has been found worldwide but is more predominant in South America. The aim of this study was to assess the presence of the RD(Rio) lineage and LAM family in the city of Rio Grande, Brazil, and to investigate the fitness of these strains based on determination of their growth rate. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different patterns were found by spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats. The predominant genotypes belonged to the LAM family (54% of the strains) followed by clade T (22%) and Haarlem (16%). The RD(Rio) lineage represented 38% of the total strains and 70.4% of the LAM strains found in this study. Strains belonging to the LAM family showed a fitness advantage when comparing their rate of growth with that of non-LAM strains, but a significant difference between RD(Rio) and non-RD(Rio) strains was not confirmed.