Knowledge of population boundaries and long-distance movements is important for wildlife conservation. We used genetic tools to investigate genetic diversity, population structure, and movements of mountain lions (Puma concolor) in Texas. We amplified 11 microsatellite loci for 245 individuals collected during 1985–2010 from Texas and New Mexico. Bayesian clustering and values of F_ST suggested a partitioning of mountain lions into 3 genetically differentiated groups, New Mexico, western Texas, and southern Texas. New Mexico and western Texas exhibited moderate levels of genetic diversity (expected heterozygosity [H_E] = 0.61 and 0.58, respectively), whereas diversity in southern Texas was lower (H_E = 0.47). Southern Texas displayed elevated genetic structure when compared to western Texas and New Mexico (F_ST = 0.102–0.148), whereas the comparison between New Mexico and western Texas revealed less subdivision (F_ST = 0.056). We documented long-distance movement among regions, and New Mexico and western Texas were sources for putative dispersers we sampled outside known populations. Differences in genetic structure and diversity between southern and western Texas support the designation of separate management units. Southern Texas appears isolated and further investigation is needed to determine the current population status. Mountain lion populations in New Mexico and western Texas may be important for future recolonization into portions of the southern United States.