The main objective of this study included sequence characterization of nuclear rDNA internal transcribed spacers ITS 2 and mitochondrial DNA CO II gene and 16 S gene fragment as potential molecular markers for studying genetic relatedness and phylogenetic kinship among five important species of genus Culex. After the extraction of genomic DNA from a single female mosquitoe followed by its amplification ITS 2 was found to be GC rich where as CO II and 16 S were AT rich. The incidence of indels was found to be maximum in ITS 2 and minimum in 16 S gene fragment. According to Maximum Parsimony based trees with Threshold (considering the trasnsversions) showed that Anopheles stephensi and Culex quinquefasciatus were supported by 100% bootstrap value in ITS 2 and 16 S gene fragment. But 100% bootstrap value was present between Anopheles stephensi and Culex bitaeniorhynchus in CO II gene. Beyond this all other species got bifurcated into two clades: one clade consisted of Culex tritaeniorhynchus + Culex mimeticus with bootstrap value of 62.8% while other clade consisted of Culex bitaeniorhynchus + Culex vishnui with bootstrap value of 19.5%.