Repeated exposure to insecticides, particularly pyrethroids and organophosphates, has resulted in the development of insecticide resistance in the mosquito Culex pipiens, a primary disease vector. Glutathione S-transferase (GST) is involved in the phase II detoxification of numerous xenobiotics, including insecticides. In this study, a GST gene (CPIJ002678) was amplified, sequenced, and used in comprehensive molecular analyses ending up in development of a rapid assay to distinguish more tolerant individuals from susceptible Culex pipiens using the Restriction Fragment Length Polymorphism (RFLP) technique. Field collected Culex pipiens strains from untreated areas, organophosphates-treated areas and a lab strain reared for many generations, all were used in CDC bottle bioassays to evaluate the susceptibility status of the studied individuals to malathion insecticide. Interestingly, both field sites collected groups showed high levels of resistance at the malathion diagnostic time. Gene amplification, and bidirectional direct sequencing results were analyzed. Compared with the reference genome sequence, the pairwise alignment of the amplified sequences showed 96.6% similarity to the reference sequence in the GenBank database. The confirmed gene sequences were assembled and aligned using various bioinformatic softwares. The assembled contigs were used in NEBcutter V2.0 for constructing restriction maps and checked for the availability of differences (if present) between susceptible and more tolerant strains. Specific molecular RFLP markers were successfully recognized to differentiate the more tolerant from the susceptible Culex pipiens phenotypes.