The nucleotide diversities and genetic relationship in the three Japanese pine species, P. thunbergii, P. densiflora, and P. luchuensis, were measured using low-copy anchor loci in Pinaceae. The average nucleotide diversity among these three Japanese pines revealed that P. thunbergii was the highest (6.05 × 10−3), followed by P. densiflora (5.27 × 10−3) and P. luchuensis (5.02 × 10−3). In comparison to other conifer species, it was concluded that the pines possessed an intermediate level of nucleotide diversity. The Heat shock protein (HSP) gene in P. thunbergii, Phenylalanine tRNA synthetase, RuBP carboxylase, and Disease resistance response protein 206 genes in P. densiflora were significantly deviated from standard neutral models. Some of these genes were related to stress or pathogen/defense response. As the samples used in this study were collected from natural populations that showed specific characteristics of being resistant to pine wilt nematode, it was hypothesized that the initial selection was an important factor in discriminating the deviation from neutrality models. Phylogenetic reconstruction revealed that the three Japanese pines were split into two lineages corresponding to P. densiflora and P. thunbergii–P. luchuensis. The latter lineage was differentiated further into two clades; P. thunbergii and P. luchuensis. The result concludes that the three Japanese pines are closely related and P. thunbergii is genetically closer to P. luchuensis, than P. densiflora.