DNA sequences of the chloroplast spacer region between thetrnP andtrnW genes (234 bps) were determined for two Japanese fir species,Abies firma Sieb. et Zucc. andA. homolepis Sieb. et Zucc., using four individuals from each species. No intraspecific variation was found in either species, but interspecific sequence polymorphism was detected between the two species. The interspecific variation was three nucleotide changes, from the 148th to 150th nucleotide position. These three nucleotides were TAC inA. firma and GTA inA. homolepis, which were inverted. In order to identify the cpDNA haplotypes between the two species, this inversion polymorphism was utilized to develop a new marker. Species-specific primers were designed so that the 3′ ends of the primers would anneal to the mutation site, in order for the two haplotypes to be easily identified by a polymerase chain reaction (PCR).