Charolais is one of the most important beef cattle breeds in the world. In Mexico, it was introduced almost a century ago, and it has been suggested that particular breeding management and genetic material origin have caused a process of divergence among the current population. By a high-density SNP array genome-wide analysis, this study aimed to assess the proposed differentiation and population structure of local populations by genetic distances and structure approaches, and a European Charolais sample was included as a reference population. The differentiation statistics indicated that local populations exhibit moderate divergence, confirming a significant differentiation process between northeastern and northwestern Charolais populations (Fst≥ 0.043, D≥ 0.031). These results were strongly supported by PCA and structure analysis. Genetic isolation and low genetic flow between populations and divergent origins of introduced genetic material (i.e., semen) are likely the main drivers of the outcomes. Some implications are discussed.