Twelve polymorphic tetranucleotide microsatellite loci were isolated and characterized for the leaf-frog Phyllomedusa burmeisteri, an endemic species of the Brazilian Atlantic forest. These loci were screened in 25 individuals from two populations of the Minas Gerais State (Carangola and Juiz de Fora). The number of alleles per locus ranged from 3 to 16 (mean = 8). Observed and expected heterozygosities ranged from 0.25 to 0.92 and 0.56 to 0.92, respectively. Evidence for both the presence of null alleles and Hardy- Weinberg equilibrium deviations were found in loci Phybu4, Phybu17, and Phybu21. Genotypic disequilibrium for each pair of loci across populations was not significant. Cross-species amplification was successful for 11 of the 12 developed loci for the sister-species, P. bahiana. These microsatellites will be important for future fine-scale population structure analyses.