Mycobacterium tuberculosis (Mtb) strains designated as RD^Rio are responsible for a large cluster of new cases of tuberculosis (TB) in Rio de Janeiro. They were previously shown to be associated with severe manifestations of TB. Here, we used three genotyping methods (IS6110 RFLP, spoligotyping, and multiplex PCR) to characterize RD^Rio and non-RD^Rio strains from the metropolitan area of Vitória, State of Espirito Santo in southeast Brazil to determine strain diversity and transmission patterns. Strains with identical IS6110 RFLP patterns were considered to belong to a cluster indicative of recent transmission. Between 2000 and 2010, we identified 5470 new TB patients and genotyped 981 Mtb strains. Of these, 376 (38%) were RD^Rio. By RFLP, 180 (48%) of 376 RD^Rio strains and 235 (40%) of 593 non-RD^Rio strains belonged to RFLP cluster pattern groups (p = 0.023). Simpson’s diversity index based on RFLP patterns was 0.96 for RD^Rio and 0.98 for non-RD^Rio strains. Thus, although RD^Rio strains appear to be comprised of a fewer number of RFLP genotypes, they represent a heterogeneous group. While TB cases caused by RD^Rio appear more likely to be due to recent transmission than cases caused by non-RD^Rio strains, the difference is small. These observations suggest that factors other than inherent biological characteristic of RD^Rio lineages are more important in determining recent transmission, and that public health measures to interrupt new transmissions need to be emphasized for TB control in Vitória.