Elusive carnivores present a challenge to managers because traditional survey methods are not suitable. We applied a genetic approach using museum specimens to examine how historical and recent conditions influenced the demographic history of Puma concolor in western and southern Texas, USA. We used 10 microsatellite loci and indexed population trends by estimating historical and recent genetic diversity, genetic differentiation and effective population size. Mountain lions in southern Texas exhibited a 9% decline in genetic diversity, whereas diversity remained stable in western Texas. Genetic differentiation between western and southern Texas was minimal historically (FST = 0.04, P < 0.01), but increased 2–2.5 times in our recent sample. An index of genetic drift for southern Texas was seven to eight times that of western Texas, presumably contributing to the current differentiation between western and southern Texas. Furthermore, southern Texas exhibited a >50% temporal decline in effective population size, whereas western Texas showed no change. Our results illustrate that population declines and genetic drift have occurred in southern Texas, likely because of contemporary habitat loss and predator control. Population monitoring may be needed to ensure the persistence of mountain lions in the southern Texas region. This study highlights the utility of sampling museum collections to examine demographic histories and inform wildlife management.