Indigenous barberry species in Spain, i.e., Berberis vulgaris subsp. seroi and B. vulgaris subsp. australis, may serve as alternate (sexual) hosts for rust pathogens infecting cereals and grasses. In this study, barberry leaves bearing aecia were collected in 2018 and 2019 from Leon, Burgos, Alava, Navarra, Huesca, Teruel, and Albacete provinces of Spain, where Berberis spp., cereal crops, and grasses coexist within certain areas. Host specificities on cereal crops and grasses of rust pathogens were investigated by pooled inoculations on varieties of wheat, barley, rye, oat, ryegrass, and cock’s-foot using aeciospores from barberry leaves. A high number of single-pustule isolates of Puccinia graminis were recovered on cereal hosts, i.e., wheat (Morocco and Line E), barley (Hiproly), rye (Prolific), oat (Marvellous), and some few on grasses, indicating that Berberis spp. are functional as alternate hosts for cereal and grass rust pathogens in Spain. DNA sequencing of aecial clusters and SSR genotyping of recovered single-pustules also confirmed that Berberis spp. may function as alternate hosts and contribute to the genetic diversity of Puccinia graminis in Spain. Stem rust infections were observed on cereal crops and grasses of the Poaceae family in close proximity to infected barberries. This indicated that the sexual cycle of P. graminis on Berberis spp. have likely contributed to stem rust infections of cereal crops and grasses and generated diverse and unique virulences of P. graminis f. sp. tritici, as observed in wheat stem rust samples in the country. Furthermore, Puccinia graminis alternating between the indigenous Berberis spp. and indigenous grasses such as Aegilops geniculata, A. triuncialis, Avena sterilis, and Helictotricon bromoides, likely represents a native and complex rust system. Further investigations into this rust pathosystem will help to understand the roles of alternate and accessory hosts in pathogen variability and disease epidemiology of rust pathogens and facilitate the development of better strategies to control rust pathogens infecting cereal crops and grasses.