Malaria remains one of the leading causes of death worldwide, particularly in Africa, where various control interventions such as case management, insecticide-treated nets (ITNs), intermittent preventive treatment in pregnant women and infants, and indoor residual spraying (IRS) have been implemented. Insecticide resistance in malaria vectors poses a major challenge to vector control efforts. This study assessed the insecticide resistance status of Anopheles gambiae s.l. populations in rice agroecosystems of Anambra State, Nigeria. Mosquito larvae were sampled from four rice-farming clusters in Anambra state, reared to adulthood at the National Arbovirus and Vector Research Center (NAVRC) and tested using the WHO susceptibility bioassay against four insecticides namely dichloro-diphenyl-trichloroethane (DDT), pirimiphos-methyl, bendiocarb, and deltamethrin. Knockdown resistance (kdr) mutations were also analyzed. Results revealed confirmed resistance to DDT and deltamethrin, likely due to past agricultural use of DDT and cross-resistance with pyrethroids, which are widely used in ITNs and IRS. However, An. gambiae s.l. populations remained susceptible to bendiocarb and pirimiphos-methyl, except in Umunze, where resistance to pirimiphos-methyl was detected. The L1014F kdr mutation was detected at varying frequencies across locations, a key genetic marker for resistance to pyrethroids and DDT, in both An. gambiae s.s. and An. coluzzii. These findings highlight the importance of continuous insecticide resistance monitoring in rice agroecosystems, improved pesticide regulations and the implementation of integrated vector control management to slow down the spread of resistance in malaria vectors in Anambra