Barberry (Berberis spp.) hosts the aecial stage of several rust species, including Puccinia graminis, which causes stem rust on grasses and cereals. The aecial stage of this pathogen has received less attention because it is not as economically important compared with the uredinial and telial stages. The main objective of this study was to identify and describe the rust species that were found on Berberis spp. collected in different parts of Sweden. A morphological study, including spore measurements and aecia descriptions, was conducted, as well as DNA sequence analyses (using the internal transcribed spacer region and the EF1-α gene). Based on spore and aecia morphology as well as the genetic analyses, three different taxa could be distinguished on barberry: P. graminis f. sp. avenae, P. graminis f. sp. tritci/secalis, and P. arrhenatheri. The genetic analysis revealed little or no differentiation between P. graminis f. sp. tritici and P. graminis f. sp. secalis and, thus, this group of samples was denominated P. graminis f. sp. tritici/secalis. Aecial morphology may be used to differentiate between different taxa. In particular, examination of aecial cross-sections may be used to distinguish between P. graminis f. sp. avenae and P. graminis f. sp. tritici/secalis. A clear differentiation in the mode of growth on barberry was also found; P. arrhenatheri always appeared systemic whereas P. graminis always appeared localized.