This study investigates species boundaries in the polyploid complex of Rosa sect. Cinnamomeae east of the Rocky Mountains. This complex is characterized by extensive intra-specific polymorphism that is the consequence, in part, of hybridization and polyploidy. An objective multivariate approach is employed to delimit species in the complex, which involved cluster and ordination analyses of 25 quantitative morphological characters and of amplified fragment length polymorphisms (AFLPs). Because polyploid individuals blurred species boundaries in the complex, they were discriminated a priori using stomata guard cell lengths in order to investigate species boundaries at each ploidy level separately. Four distinct species were found at the diploid level: R. blanda – R. woodsii, R. foliolosa, R. nitida and R. palustris. According to the morphological and molecular data, R. blanda and R. woodsii are indistinguishable and should be considered as a single species. Three species were identified at the polyploid level, R. arkansana, R. carolina, and R. virginiana, albeit with evidence of hybridization between them. The genetic and morphological similarity between individuals of the polyploid species and those of the different diploid species allowed us to identify possible parents for the polyploid species. Rosa arkansana likely originated from R. blanda (incl. R. woodsii), R. carolina from a hybrid between R. blanda and R. palustris, and R. virginiana from R. palustris. Although the multivariate approach was not able to differentiate species when all individuals were considered together, a classification tree showed that it is indeed feasible to identify species in the complex without prior knowledge of the ploidy level of individuals.