Pumas Puma concolor are one of the most studied terrestrial carnivores because of their widespread distribution, substantial ecological impacts, and conflicts with humans. Over the past decade, managing pumas has involved extensive efforts including the use of genetic methods. Microsatellites have been the most commonly used genetic markers; however, technical artifacts and little overlap of frequently used loci render large-scale comparison of puma genetic data across studies challenging. Therefore, a panel of genetic markers that can produce consistent genotypes across studies without the need for extensive calibrations is essential for range-wide genetic management of puma populations. Here, we describe the development of PumaPlex, a high-throughput assay to genotype 25 single nucleotide polymorphisms in pumas. We validated PumaPlex in 748 North American pumas Puma concolor couguar, and demonstrated its ability to generate reproducible genotypes and accurately identify individuals. Furthermore, in a test using fecal deoxyribonucleic acid (DNA) samples, we found that PumaPlex produced significantly more genotypes with fewer errors than 12 microsatellite loci, 8 of which are commonly used. Our results demonstrate that PumaPlex is a valuable tool for the genetic monitoring and management of North American puma populations. Given the analytical simplicity, reproducibility, and high-throughput capability of single nucleotide polymorphisms, PumaPlex provides a standard panel of markers that promotes the comparison of genotypes across studies and independent of the genotyping technology used.