Twenty-four dinucleotide simple sequence repeat markers were developed for the phytopathogenic fungus, Puccinia graminis. The identified loci were polymorphic, with allelic diversity ranging from two to 11 alleles. Observed and expected levels of heterozygosity ranged from 0.000 to 0.960 and from 0.113 to 0.846, respectively. Fourteen of the loci deviated significantly from Hardy–Weinberg equilibrium. Null alleles were observed for 10 of the 24 loci with a frequency of 4–16%. A preliminary screen of other Puccinia cereal rust fungi (P. coronata, P. striiformis and P. triticina) indicated that these primer pairs are specific to P. graminis.