Litoria nannotis is an endangered waterfall frog from the wet tropics region in north Queensland that has suffered significant population declines due to the emerging fungal disease known as chytridiomycosis. The species has two deeply divergent lineages, and we used 454 shotgun sequencing of DNA extracted from one individual of the northern lineage to identify and design PCR primers for 576 microsatellite loci. Thirty markers were tested for amplification success and variability in a population sample from each lineage. Of these, 17 were found to be polymorphic in the northern lineage and 10 loci were polymorphic in the southern lineage. Numbers of alleles per locus ranged from 2 to 14 (mean = 6.47, s.d. = 4.02) for the northern lineage (17 polymorphic loci), and from 2 to 8 (mean = 5.40, s.d. = 2.55) in the southern lineage (10 polymorphic loci). Levels of heterozygosity were high in both lineages (northern mean H_E = 0.63, s.d. = 0.21, range = 0.27–0.89; southern mean H_E = 0.57, s.d. = 0.25, range = 0.18–0.81). These loci will be useful in understanding the genetic variation and connectivity amongst populations of this species recovering from mass population declines due to disease.