During 2012-2013, yellow rust samples from barley, wild barley (Hordeum morinum) and wheat from different provinces of Iran were used to inoculate Afzal (barley cultivar) and Morocco (wheat cultivar), susceptible to all Puccinia striiformis f. sp. hordei (Psh) and Puccinia striiformis f. sp. tritici (Pst) isolates, respectively. The isolates were identified as Psh or Pst based on virulence on barley or wheat differential seedlings, respectively (Chen et al., 1995). In order to study of genetic diversity, we used 18 isolates representative of different pathotypes from different hosts. The genetic diversity was assessed using five simple sequence repeat (SSR) markers and by examining intergenic spacer 1 (IGS1) region. A total of 14 genotypes were identified within the studied isolates, of which eight genotypes related to Psh and six genotypes related to Pst. The multiplied fragments of some SSR loci, in particular RJ2N, showed that some isolates have alleles in common with isolates of Psh and Pst. Dendrograms were generated using virulence and markers data. Clustering analysis showed groups related to their hosts: isolates infecting only wheat, isolates infecting only barley; isolates infecting both wheat and barley differential genotypes; and isolates infecting only wild grasses with some overlaps. The groups infecting both barley and wheat differentials (based on virulence data) and having some SSR alleles in common with Psh and Pst, appeared to be hybrid group between two formae speciales. Before this report, Cheng and Chen (2009) and Holtz et al. (2014) had reported hybridization between two formae speciales.