Microsatellite loci specifically designed and optimized for secondary vectors and neglected species of mosquitoes are lacking. Cross-species transferability of microsatellite loci between close taxa has been used as a reliable option, being successfully done for Aedes taeniorhynchus Wiedemann (Diptera: Culicidae), Triatoma sordida Stål (Hemiptera: Reduviidae), and Aedes fluviatilis Lutz (Diptera: Culicidae). Nonetheless, prior to the utilization of transferred microsatellites, they must be assessed for their performance and reliability on the target species. Therefore, the goal of this study was to transfer and validate SSR primers previously developed for the Culex pipiens complex to Culex (Culex) nigripalpus Theobald (Diptera: Culicidae). Culex nigripalpus were collected in seven different locations throughout the city of São Paulo using CDC traps baited with dry ice between 2011 and 2013, yielding a total of 223 specimens. Six SSR primers were successfully transferred to Cx. nigripalpus yielding satisfactory results for allele richness, ranging from 5 to 12 alleles per locus. The expected heterozygosity values were higher than the observed heterozygosity (P < 0.05) for the loci CxqCTG10, CxqGT51, and Cxpq59. Linkage disequilibrium results were statistically significant only for the comparison between the loci CxqCA115 and CxqCTG10, and the estimation for the probability of null alleles was also low, ranging from 0 to 0.052.These results substantially validate the SSRs transference to Cx. nigripalpus as well as their subsequent use in microevolution studies comprising this species.