The ecology of a species strongly influences genetic variation and population structure. This interaction has important conservation implications because taxa with low dispersal capability and inability to use different habitats are more susceptible to anthropogenic stressors. Ocelots (Leopardus pardalisalbescens) and bobcats (Lynx rufus texensis) are sympatric in Texas and northeastern Mexico; however, their ecology and conservation status are markedly different. We used 10 microsatellite loci and a 397-bp segment of the mitochondrial control region to examine how historical and ecological differences in these two species have influenced current patterns of genetic diversity in a landscape heavily altered by anthropogenic activities. Substantially higher genetic diversity (heterozygosity and haplotype diversity) and population connectivity was observed for bobcats in comparison to ocelots. The level of divergence among proximate ocelot populations (<30 km) was greater than between bobcat populations separated by >100 km. Ocelot populations in the US have never recovered from reductions experienced during the twentieth century, and their low genetic variation and substantial isolation are exacerbated by strong preference for dense native thornshrub and avoidance of open habitat. In contrast, despite continued legal harvesting and frequent road-related mortality, bobcats have maintained wide distribution, high abundance, and population connectivity. Our study illustrates that sympatric species with a similar niche can still have sufficient ecological differences to alter their response to anthropogenic change. Sensitive species, such as the ocelot, require additional conservation actions to sustain populations. Ecological differences among species occupying a similar guild are important to consider when developing conservation plans.