Subalpine larch (Larix lyallii Parl.) and western larch (Larix occidentalis Nutt.) represent two closely related species with contrasting abundance and distribution patterns in Western North America. Genetic diversity at seven informative microsatellite loci was determined for 19 populations of subalpine larch and nine populations of western larch. Contrasting genetic diversity and patterns of population differentiation were observed between the two species. The overall within-population genetic diversity parameters were lower in subalpine larch (A = 3.2; AP = 3.6; HE = 0.418) than in western larch (AP = 5.51; HE = 0.580), a pattern that is likely related to historical or demographic factors. No evidence of interspecific hybridization was observed. Significantly more population differentiation (θ = 0.15; RST = 0.07), consistent with more restricted gene flow, was observed for subalpine larch as compared to western larch (θ = 0.05; RST = 0.04). Under the assumption of an infinite allele mutation model, 12 of the 19 subalpine larch populations showed signs of deviation from the mutation–drift equilibrium, which suggests Holocene population bottlenecks and fluctuations in effective population size for this species. None of the western larch populations deviated significantly from the mutation–drift equilibrium. For both species, Mantel's test revealed a significant positive relationship between geographical and genetic distances indicative of isolation by distance. A similar geographical structure was detected in both species, suggesting at least two genetically distinct glacial populations in each species. The various implications for gene conservation are discussed.