The high tuberculosis (TB) incidence rates, the closeness of the cities and the high migration flux on the Brazil/Paraguay/Argentina border deserves an in-depth study, using Mycobacterial Interspersed Repetitive Unit (MIRU) and Spoligotyping genetic markers to explore the impact of the Mycobacterium tuberculosis RD^Rio lineage on disease transmission and resistance to anti-TB drugs in this setting. Although without the totality of M. tuberculosis isolates causing TB in this studied setting, a number of 97 isolates obtained from sputa samples culture of patients with confirmed TB, from 2013 to 2015, were submitted to 24 loci MIRU, Spoligotyping, detection of RD^Rio lineage and detection of mutation related to isoniazid and rifampicin resistance by MTBDRplus/DNA STRIP. In this sample, it was observed high clonal variability of circulating M. tuberculosis isolates causing TB in Brazilian cities bordering Paraguay and Argentina. The percentage of RD^Rio lineage causing TB in this setting was 15.46%, and lower than the detected in different areas of Brazil. According to 24 loci MIRU, the major MIRU International Type (MIT) related with RD^Rio lineage were MIT 26, MIT 738, MIT 601 with four, two and one isolates, respectively. Eight isolates with RD^Rio marker were classified as orphans. The mainly Spoligofamily related with RD^Rio lineage was LAM1 and LAM9 and no relationship between RD^Rio lineage and resistance in M. tuberculosis isolates circulating in this setting could be established. This work is pioneer in studying the dynamics of RD^Rio lineage transmission on the Brazil/Paraguay/Argentina border and deserves further studies to analyze the real contribution of the RD^Rio lineage in outbreaks and the risk of significant development of MDR-TB in the setting studied.