Western burrowing owls (Athene cunicularia hypugaea) are ground-dwelling owls distributed throughout western North America. Because of population declines, this species is considered endangered in Canada, and burrowing owls are listed as a species of conservation concern in states of the western USA. Korfanta et al. (2002) previously presented primers for seven microsatellite loci in burrowing owls. Parentage and relatedness studies require a larger number of markers for accuracy and precision. Here, we developed and characterized 18 additional microsatellite DNA loci, and we tested these loci in 23 individuals. The number of alleles per locus ranged from 2 to 11; two loci deviated from Hardy–Weinberg equilibrium following Bonferroni correction; we did not detect linkage disequilibrium following Bonferroni correction; and the probability of exclusion for parent pairs using all loci was >0.9999. We envision these loci will facilitate detailed analyses of the genetic mating system of burrowing owls, which is poorly understood.