We developed 21 microsatellite (13 dinucleotide and 8 tetranucleotide) primers specifically for pumas (Puma concolor). The primers were tested across 243 individuals from California and Nevada, and displayed an average of 5.5 alleles per locus. Previously, domestic cat (Felis catus) primers have been adapted for puma genetic studies. Puma-specific loci may reduce concerns associated with ascertainment bias, improve genetic structure resolution and provide additional tetranucleotide loci valuable for forensic applications. These puma-specific microsatellites will aid studies involving parentage, kinship and population structure, along with forensic case applications including poaching and puma-associated injuries to humans and domestic animals.