Species ecology significantly influences genetic variation and population structure, impacting conservation. We examined genetic diversity in sympatric ocelots (Leopardus pardalis albescens) and bobcats (Lynx rufus texensis) in Texas and northeastern Mexico using microsatellite loci and mitochondrial DNA. Bobcats exhibited greater genetic diversity and population connectivity than ocelots, whose populations remain fragmented and genetically isolated due to their strong preference for dense thornshrub and avoidance of open habitats. In contrast, bobcats, despite legal harvesting and road mortality, maintain high abundance and connectivity. Our findings highlight the need for targeted conservation efforts for sensitive species like the ocelot and underscore the importance of considering ecological differences in conservation planning.