Culex tarsalis Coquillett (Diptera: Culicidae) is a highly efficient arbovirus vector. Spatial and temporal heterogeneity have been observed in Cx tarsalis for phenotypic traits including autogeny, virus susceptibility and host preference. Genetic differences between populations may in part explain these observations. Using the M13-tailed primer method, we identified 45 novel polymorphic microsatellite markers from microsatellite-enriched Cx tarsalis genomic libraries. The M13-tailed primer method was more efficient in identifying useful loci than traditional screening by acrylamide gel electrophoresis. These markers will be useful for investigating genetic questions in this important vector mosquito.