Cleavage amplified polymorphic sequence (CAPS) markers of strawberry (Fragaria x ananassa Duch.) can be useful for identifying mislabeled or patent-infringing cultivars in the marketplace. However, CAPS markers in octoploid strawberry tend to give unclear bands because multiple homologous sites are simultaneously amplified by the non-selective PCR. To overcome this problem, we used "cluster-specific amplification" based on the nucleotide sequences of PCR products and were able to improve the band clarity of 18 CAPS markers. By analyzing the marker segregation ratio, we demonstrated that 13 clarified markers were derived from single diploid loci that were transmitted to progeny in a manner consistent with Mendelian inheritance. We discuss the genomic structure of octoploid strawberry from the viewpoint of cluster and segregation analysis and suggest that it comprises independent genomes. We tested the utility of all of the markers we developed for cultivar identification and confirmed their ability to distinguish among 64 strawberry cultivars.