Mycobacterium bovis is a causal agent of bovine tuberculosis (bTB), one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of M. bovis infections of livestock is an important tool for understanding the epidemiology of bTB and defining control/eradication strategies. In this study, whole genome sequencing (WGS) of 74 M. bovis isolates sourced from naturally infected cattle in the State of Rio Grande do Sul (RS), southern Brazil, was used to evaluate the population structure of M. bovis in the region, identify potential transmission events and date the introduction of clonal complex (CC) European 2 (Eu2). In silico spoligotyping identified 11 distinct patterns including four new profiles and two CCs, European 1 (Eu1) and Eu2. The analyses revealed a high level of genetic diversity in the majority of herds and identified putative transmis sion clusters that suggested that within- and between-herd transmission is occurring in RS. In addition, a comparison with other published M. bovis isolates from Argentina, Brazil, Paraguay and Uruguay demonstrated some evidence for a possible cross-border transmission of CC Eu1 into RS from Uruguay or Argentina. An estimated date for the introduction of CC Eu2 into RS in the middle of the 19th century correlated with the historical introduction of cattle into RS to improve existing local breeds. These findings contribute to the understanding of the population structure of M. bovis in southern Brazil and highlight the poten tial of WGS in surveillance and helping to identify bTB transmission.