Long-term geographic isolation and breeding programs both influence population characteristics. Leiqiong cattle, a native breed from the southernmost region of China, are renowned for disease and heat resistance, with two subgroups on Hainan Island and the Leizhou Peninsula. However, the genomic differences between them remain unexplored. In this study, we conducted genomic comparisons using whole-genome sequencing data from the two subgroups of Leiqiong cattle and three commercial breeds to assess their population structures. Leiqiong cattle in Hainan exhibited lower genetic diversity and a pure ancestral content due to their isolation from the mainland. In contrast, the subgroup in Guangdong displayed higher genetic diversity and mixed ancestry, influenced by the intrusion of commercial breeds. The genetic divergence between them was evaluated by estimating a genetic distance of 0.08 and a split time of 3400 to 4250 years ago, highlighting the role of geographical barriers in speciation. Notably, two candidate genes were identified through selection sweeps, including