Eighteen polymorphic di- and trinucleotide simple sequence repeat markers were developed for the phytopathogenic rust fungus Puccinia triticina. The allelic diversity varied from two to nine alleles per locus. Levels of observed heterozygosity ranged from 0.095 to 0.952. Seven of the loci deviated significantly from Hardy–Weinberg equilibrium (P < 0.002) with 70% having levels of observed heterozygosity higher than expected heterozygosity. Null allele(s) were observed for locus PtSSR76 with a frequency of 9%. A preliminary screen of other cereal rust fungi (P. coronata, P. graminis, P. recondita and P. striiformis) indicated that these primer pairs are specific to P. triticina.