The spatial patterns of non-neutral genetic variations at fine spatial scales and their possible associations with microenvironments have not been well-documented for tree populations. Based on 25-32 SNP markers, we examine whether non-neutral SNPs and their associations with microenvironments can be detected in FcMYB1603, a gene homologous to that encoding a protein induced by drought stress in Arabidopsis thaliana for the 166 adult trees in a 1-ha plot in a mature population of Fagus crenata. In the 83 individuals of a younger cohort of below canopy trees, the nonsynonymous SNP at locus FcMYB1603_684 exhibited a spatial signature representing a departure from the expected spatial patterns of neutral genetic variation. Evaluations of non-neutrality for this locus were robust against the potential risks of false positives due to the low number of SNP loci, a low criterion set for minor allele frequency, and any edge effect on the trees' spatial structure. An older cohort exhibited no signal of the existence of non-neutral genetic variation, suggesting that temporal fluctuation in the microenvironmental conditions on the forest floor may have exposed different cohorts to different magnitudes of selection pressure. Although genotypes of the locus showed a spatial association with a microenvironmental variable potentially related to soil moisture, the present study was subject to a limitation due to the generally low polymorphism of nonsynonymous loci within the single plot, which suggests that it will be important to replicate the study design in order to carry out research on fine-scale non-neutral genetic variations.