454 shotgun sequencing was used to identify thousands of in silico microsatellite loci from two Australian freshwater turtle species (Pleurodira, Chelidae), Elseya albagula and Emydura macquarii krefftii. Twenty-nine polymorphic microsatellites were subsequently developed and tested as markers, 15 for E. m. krefftii and 14 for E. albagula. The former marker set showed greater allelic richness (mean N A = 7.7, range 3-15) than the latter (mean N A = 4.4, range 2-8). Levels of expected heterozygosity were high for both marker suites (mean H E = 0.718, range 0.177-0.875; and mean H E = 0.567, range 0.317-0.827, respectively), indicating both are suitable for studies of population genetic structure, connectivity and mating systems in these taxa. Cross-species amplification in 12 other short-necked freshwater turtle taxa highlighted the potential utility of these markers in other species, notably Emydura spp., Rheodytes leukops and Elusor macrurus.