An entomological survey conducted in 2002 in Guinea Bissau aimed i) to study the distribution of the members of Anopheles gambiae Giles complex (Diptera: Culicidae) throughout four ecological areas extended from mangrove to savannah ii) to evaluate the insecticide susceptibility status of these malaria vectors exposed to permethrin 0.75% and DDT4%, and finally iii) to investigate the occurrence and the spread of the Leu-Phe knock down resistance (kdr) gene associated with pyrethroid and DDT resistance within these vector populations. Adult female mosquitoes issued from indoor morning collections were tested using WHO procedures, test kits and impregnated papers to assess their insecticide susceptibility status. Tested specimens were identified by PCR assays and characterized for the kdr gene. Malaria vectors were mainly dominated elsewhere by An. gambiae s.s. (both S and M molecular forms) living in sympatry with low proportion of An. melas in the littoral. An. gambiae s.s. tested populations were fully susceptible both to permethrin 0.75% and to DDT 4% irrespective to their location and ecotypes. The Leu-Phe kdr mutation was detected at low frequency only in two sites respectively urban (Bissau) and Guinea-savannah (Gabu) areas. It occurred only in the S molecular form in Gabu (at the frequency of 0.14) and both in the S and M molecular forms in Bissau at the frequency of 0.06 and 0.02 respectively. These results suggested that the populations of An. gambiae s.s., the most frequent malaria vector in Guinea Bissau, still remain cross-susceptible to pyrethroids and DDT This susceptibility status and the frequency of resistance mechanism such as the kdr mutation must be monitored in the future particularly in the urban and savannah areas with continuous and intensive use of insecticides.