It has been attempted to develop multiple resistant rootstock against rose root rot (Pythium helicoides Drechsler) and crown gall disease (Rhizobium radiobacter (Beijerinck and van Delden, 1902) Young et al., 2001) using tetraploid Rosa multiflora 'Matsushima No.3' and R. 'PEKcougel'. However, hybrid identification based on conventional morphological characteristics is difficult, and establishment of hybrid diagnosis by DNA markers is desired. In this study, we attempted to develop specific DNA markers of R. multiflora. Three hundred 10 mer random oligonucleotide primers were screened, and a unique polymorphic fragment in R. multiflora amplified by OPAK16 primer was identified. This fragment could distinguish between roses with cross-fertilization genealogy with R. multiflora and roses with no relationship with R. multiflora. This specific fragment was cloned, sequenced and converted into a sequence characterized amplified region (SCAR) marker-SCAK16, which can amplify a 583 base pair (bp) fragment only in roses with relatedness to R. multiflora. This result indicated that SCAK16583 was a species-specific marker of R. multiflora that could be used in a marker-assisted breeding program for hybrid identification of R. multiflora. © JSHS 2013.