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Genetic variation of 16S rRNA gene sequences were investigated in two Japanese lucanid beetles, Ceruchus lignarius and Dorcus rectus rectus, which have different habitats and distributional patterns. Present results indicate that the inter-populational sequence variation of 16S rRNA within three subspecies of C. lignarius are much smaller than those between all combinations of different subspecies, suggesting the monophylies of these three subspecies. Our results also suggest that sequence divergence within a single population is very low in this species and that differentiations increase in the order of intra-population (0-0.11%), intra-subspecies (0-2.26%), intra-species (2.80-7.99%) and interspecies (7.34-10.05%). Likewise, intra-subspecific nucleotide replacements of D.r. rectus (0-1.06%) were much smaller than the interspecific divergence (14.82-19.95%).