Previous studies on Culex pipiens pipiens mosquitoes collected from Tunisia between 1990 and 1996 revealed an alarming resistance to organophosphate insecticides. However, since then very few studies have assessed the evolution of insecticides resistance in this species of mosquitoes. Three populations of Cx. pipiens pipiens mosquitoes were collected in three districts across the country between 2003 and 2005 and subjected to evaluation of their fenitrothion resistance/susceptibility status and associated mechanisms. Studied populations showed moderate resistance to the used insecticide which ranged from 7.59 to 11.8. The use of synergists revealed the lack of detoxification enzymes as metabolic resistance. However, starch gel electrophoresis indicated that resistance at the Ester locus was attributed to different Ester alleles. On the other hand, the positive cross-resistance to organophosphate fenitrothion and carbamate propoxur insecticides indicating a high prevalence of insensitive AChE1 mechanism in field populations. AChE activity showed that all populations exhibited a significant excess of heterozygotes ([RS]) compared to other detected genotypes ([SS] and [RR]) in the studied samples. Our analysis suggests that the heterozygotes often have a fitness advantage over the homozygotes.