Genetic structure of a population can be molded by the resistance of the landscape or the distance between populations that function as barriers to gene flow. We analyzed the population genetic structure of Abies religiosa on a fine spatial scale and examined isolation models by resistance and distance. We collected vegetative tissue from populations located at the altitudinal extremes of the distribution range of the species on three slopes of La Malinche National Park (LMNP) (South, North, and East) in central Mexico. Genomic DNA was obtained using the CTAB 2X method, and eight microsatellite chloroplast loci were amplified. The genetic structure was identified based on an Analysis of Molecular Variance, a Discriminant Analysis of Principal Components with cross-validation and a spatial Principal Component Analysis using the Gabriel-type connectivity network. The isolation hypotheses were evaluated by constructing partial Mantel tests using Reciprocal Causal Modeling and Maximum Likelihood Population Effects models. A genetic structure of isolation by resistance to elevation was identified, and two genetic groups were recognized: one including populations of the South slope and the other comprising populations of the North and East slopes. We detected in Abies religiosa populations of the LMNP an isolation by resistance to elevation that determines the genetic structure, and the greatest genetic exchange between groups of populations located at higher altitudes. It is suggested to promote the connectivity between slopes through assisted migration and immediately halt land-use changes, as part of the actions to preserve genetic diversity in the LMPN. This study contributes to the knowledge of the spatial genetic structure of species at risk in the Mexican temperate forest for their conservation.