Microsatellite markers were isolated and developed from Culex pipiens quinquefasciatus Say (Diptera: Culicidae) sampled in Johannesburg, South Africa, to identify those that are broadly useful for analyzing Cx. pipiens complex populations between continents. Suitable loci should be 1) inherited in a codominant Mendelian manner, 2) polymorphic, 3) selectively neutral, 4) randomly associated, 5) without null alleles, and 6) applicable across broad regions and between diverse biotypes. Loci in Cx. p. quinquefasciatus from Johannesburg ranged from two to 17 alleles per locus and expected heterozygosities (H_e) were 0.02–0.87. Loci in Cx. p. pipiens L. from Johannesburg had five to 19 alleles per locus and H_e values ranging from 0.57 to 0.93, whereas those from George, South Africa, had five to 17 alleles per locus and H_e values ranging from 0.54 to 0.88. Loci in North American mosquitoes were more variable. Cx. p. quinquefasciatus from South Carolina had five to 19 alleles per locus and H_e values ranging from 0.64 to 0.90, whereas Cx. p. pipiens from Massachusetts had six to 28 alleles per locus and with H_e values ranging from 0.65 to 0.94. All loci were associated randomly. Overall, four of nine of these new loci satisfied all six criteria for broad utility for analyzing the genetic structure of Cx. pipiens populations.