Ecotypic divergence in tree taxa often occurs in sub-alpine habitats, where environmental conditions are more stressful than those in lower elevations. In the mountain oak species in Japan, Quercus crispula (Qc), the sub-alpine shrubby variety, Q. crispula var. horikawae (Qch), has been recognized in central and northern Honshu. Although Qch has different phenotypes from Qc, genetic divergence between Qc and Qch has not been examined yet. Pairs of Qc and Qch populations in eight locations and additional Qc and Qch populations around these locations were investigated. Leaf size of Qch was smaller than that of Qc. Chloroplast DNA haplotypes were shared between the Qc and Qch populations. In genotypes at 29 nuclear microsatellite loci, genetic diversity did not differ between the Qc and Qch populations. Principal component analysis and a neighbor-joining tree of populations based on microsatellite genotypes demonstrated that 13 Qc populations and eight Qch populations were grouped separately, except for three Qch populations that were grouped to Qc. Climatic conditions in the eight Qch populations were characterized by lower temperature and heavier snowfall than those in the 16 populations of the genetic group of Qc. These results suggest the genetic divergence between Qc and Qch associated with sub-alpine climatic conditions, irrespective of leaf size. The origin of the sub-alpine Qch lineage and the history of ecotypic divergence should be investigated in future genomic studies.