The Pearl Darter Percina aurora is an imperiled species of fish currently only found in the Pascagoula River drainage in southern Mississippi. We tested 60 microsatellite loci identified by Illumina pair-ended sequencing. Forty of these loci were polymorphic in Pearl Darters with a mean number of alleles per locus of 11.5 and mean observed and expected heterozygosity values of 0.818 and 0.805, respectively. Eleven to 17 of these loci were also polymorphic in the closely related Channel Darter P. copelandi and Coal Darter P. brevicauda. These loci should prove useful in genetic studies associated with informing future management decisions for Pearl Darters and conservation efforts in other species of darter.