Malaria, transmitted by infected female Anopheles mosquitoes, is a parasitic disease of major public health importance in Nigeria. Various classes of insecticides have played significant role in the control and elimination of malaria by attacking the vectors. Unfortunately, the success of the use of insecticides in the control of malaria vectors is threatened by the emergence of insecticidal resistance. This study was therefore aimed at determining the resistance status of Anopheles gambiae s. l. in communities in Anambra State, southeast Nigeria. Larval samples were collected from diverse habitats and reared to adult for susceptibility test. Target site resistance assays were carried out and genotypic differentiation test was performed to assess variability of the allelic frequencies of the kdr (L1014F) mutation. The results showed that Anopheles gambiae was the predominant vector (54.2%), followed by Anopheles coluzzii (45.8%). The distribution of the frequency of resistant allele of the kdr gene F (L1014F) showed that the number of homozygous resistant genotype (RR) in Awka North LGA for An. gambiae and An. coluzzii species was highest (31), followed by Awka South (20), and Njikoka (5) LGAs. Also, the number of Heterozygous resistant genotype (Rr) was highest in Awka South (7), followed by Awka North (2), and none was recorded in Njikoka LGA. Homozygous resistant genotype (RR) was frequently observed in the three study areas of Anambra State and is a threat to the use of pyrethroids for insecticidal control of malaria vectors. There is an urgent need for implementation of insecticide resistance management and monitoring in Anambra State to assess the spread of resistance which may expand malaria vector resistance to the remaining classes of insecticides (organophosphates and carbamates) on the long run.