The amount and structure of the genetic diversity in Mexican populations of Pseudotsuga menziesii (Mirb.) Franco, is almost unknown, since most genetic studies have been carried out on populations from Canada and the United States. Here, we applied a set of 12 microsatellite markers to 12 populations (234 trees) from the central region of Mexico in order to determine values of genetic diversity and differentiation. Seventy-three different alleles were identified: an average number of alleles per locus (Na) of 6.083, effective number of alleles (Ne) of 2.039, observed heterozygosity (Ho) of 0.229, and expected heterozygosity (Ht) of 0.417. Genetic differentiation was high: the coefficient of differentiation (θ) was 0.270, while the coefficient of structure (Φ_st) was 0.278. Bayesian analysis identified two genetic groups in central Mexico. The PCoA and the dendrogram were in concordance with the two genetic groups. The results of the analysis of molecular variance (AMOVA) indicate that genetic variation exists mainly within populations (72.149%). Therefore, conservation efforts should focus on as many individuals within populations as possible, to maintain this variation.