We investigated 25 natural populations of Chamaecyparis obtusa using 51 cleaved amplified polymorphic sequence (CAPS) markers, which were developed using information on sequence-tagged sites (STS) in Cryptomeria japonica. Most CAPS markers have codominant expression patterns, and are suitable for population studies because of their robustness and convenience. We estimated various genetic diversity parameters, including average heterozygosity (H e ) and allelic richness and found that the more peripheral populations tended to have lower genetic diversity than central populations, in agreement with a previous theoretical study. The overall genetic differentiation between populations was low, but statistically significant (G ST ¼ 0.039), and similar to the level reported in a previous allozyme study. We attempted to detect non-neutral loci associated with local adaptation to clarify the relationship between the fixation index (F ST ) and H e values for each locus and found seven candidates non-neutral loci. Phylogenetic tree analysis of the populations and Bayesian clustering analysis revealed a pattern of gradually increasing isolation of populations with increasing geographical distance. Three populations had a high degree of linkage disequilibrium, which we attribute to severe bottlenecks due to human disturbance or competition with other species during their migration from refugia after the most recent glaciation. We concluded that the small populations in western Japan and in Kanto district are more important, from a conservation perspective, than the populations in central Japan, due to their genetic divergence, relatively small sizes and restricted areas.