Pinus aristata Engelm., Rocky Mountain bristlecone pine, has a narrow core geographic and elevational distribution, occurs in disjunct populations, and is threatened by rapid climate change, white pine blister rust, and bark beetles. Knowledge of genetic diversity and population structure will help guide gene conservation strategies for this species. Sixteen sites across four mountain ranges in the core distribution of P. aristata were sampled and genetic diversity was assessed with 21 isozyme loci. Low species and population level genetic diversity (He = 0.070 and 0.062, respectively) occurred with moderate among-population differentiation (FST = 0.131). Genetic diversity correlated with longitude, latitude, and elevation and a strong mountain island effect may contribute to substructuring and isolation. Using multiple complementary analyses, sampled trees were assigned to three genetic lineages that varied in diversity and admixture and were associated with different climatic factors. The distribution of genetic diversity and substructuring of P. aristata may be an outcome of a combination of restricted gene flow due to geographic and phenological isolation, random processes of genetic drift, life history traits, natural selection, and postglacial migrations. The combination of low genetic diversity, moderate population isolation, and a protracted regeneration dynamic puts populations at risk for extirpation by novel stresses.