Here, we describe the development of seven new microsatellite loci from Petaurus breviceps. Together with eight loci from previous studies of gliders, we tested their utility for amplification, multiplexing and polymorphism in two glider species, P. breviceps and P. norfolcensis. Of the 15 loci tested, all were polymorphic in P. breviceps and 12 were polymorphic in P. norfolcensis. Overall, 260 sugar gliders from 13 sites in south-eastern South Australia and 106 squirrel gliders collected throughout south-east Queensland were used in analyses. Numbers of alleles per locus ranged from 4 to 27 in P. breviceps and from 2 to 44 in P. norfolcensis. Observed heterozygosity ranged between 0.438 and 0.904 in P. breviceps and between 0.189 and 0.981 in P. norfolcensis. Within the populations analysed, one of the 15 loci for P. breviceps and two of the 12 loci for P. norfolcensis deviated from Hardy–Weinberg equilibrium. The microsatellite loci will provide valuable tools for further study of social organisation, mating systems and population biology of these gliding marsupials.