Sex defining loci or chromosomes often contain large non-recombining regions. In Basidiomycota, two distinct gene loci confer mate compatibility. These loci encode for homeodomain (HD) transcription factors and pheromone receptor (PR)-ligand pairs. In some fungi these two loci are physically linked and give rise to large evolutionary strata with extensive recombination suppression. To date genome level mating type (MAT) loci analysis is lacking for obligate biotrophic basidiomycetes that depend on their host plants for survival, this includes the orderPucciniales. These economically important plant pathogens have long-term dikaryotic life-stages in which matched haploid nuclei must carry compatibleMATloci.Here we focus on fourPucciniacereal rust species, includingPuccinia graminisf.sptritici,P. striiformisf.sptritici,P. triticinaandP. coronataf.spavenae, which infect major cereals including wheat and oat. We identifiedMATlocations on two separate chromosomes that supports previous hypotheses of a tetrapolar mating system in thePucciniales.HDloci displayed largely conserved macrosynteny among species and were not suppressed for recombination. Consistently,HDtranscription factor genes showed no trans-specific polymorphisms between species. Regions, surrounding thePRloci however, displayed extensive genomic degeneration, signs of recombination suppression and were found to be linked to centromeres in some species. Expression analysis suggests that bothMATloci are involved in nuclear pairing during spore formation in the asexual cycle. Together, our study provides insights into the evolution ofMATloci since speciation of key pathogenicPucciniaspecies. This detailed understanding is important to predict the possible combinations of nuclear pairs that can arise via sexual reproduction, somatic recombination, or di-om mating to enable the evolution of newly virulent isolates of these agriculturally important plant pathogens.