We used three different methods, size-selected genomic library, cross-species amplification of a mammal-wide set of conserved microsatellites and genomic sequencing, to develop a panel of 43 microsatellite loci for the short-beaked echidna (Tachyglossus aculeatus). These loci were screened against 13 individuals from three different regions (Tasmania, Kangaroo Island, Perth region), spanning the breadth of the range of the short-beaked echidna. Nine of the 43 tested loci amplified reliably, generated clear peaks on the electropherogram and were polymorphic, with the number of alleles per locus ranging from two to eight (mean = 3.78) in the individuals tested. Polymorphic information content ranged from 0.16 to 0.78, and expected heterozygosity ranged from 0.19 to 0.84. One of the nine microsatellites showed a heterozygote deficit, suggesting a high probability of null alleles. The genomic sequencing approach using data derived from the Roche FLX platform is likely to provide the most promising method to develop echidna microsatellites. The microsatellite markers developed here will be useful tools to study population genetic structure, gene flow, kinship and parentage in Tachyglossus sp. and potentially also in endangered Zaglossus species.