In Tunisia, the mosquito Culex pipiens shows various organophosphate resistance alleles at Ester and ace-1 loci. The characterization and the distribution pattern of these alleles were studied among 20 populations sampled from north to center of Tunisia. At the Ester locus, Ester⁴, Ester⁵, and Ester^B12 were present. A new esterase characterized by the same electrophoretic migration as esterase Al was identified: A13, encoded by EsterA¹³ allele. At the ace-1 locus, the presence of the ace-1^R, ace-1^D, and F290V mutated alleles was also detected. A large heterogeneity in allelic frequencies at Ester and ace-1 loci was observed among samples, with a high significant genotypic differentiation considering both loci (F_st = 0.077, P <10^-5), depicting variations of insecticide treatment intensity between areas. A comparison between populations collected in 1996 and 2005 showed an absence of significant resistance evolution. However, the high frequencies of resistance alleles in 2005 populations suggested that the selection pressures are still important in Tunisia. Strategies for resistance management are discussed in the context of the current knowledge of the Tunisian situation.