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Background: The emergence and spread of insecticide resistance in malaria vectors to major classes of insecticides call for urgent innovation and application of insecticides with novel modes of action. When evaluating new insecticides for public health, potential candidates need to be screened against both susceptible and resistant mosquitoes to determine efficacy and to identify potential cross-resistance to insecticides currently used for mosquito control. The challenges and lessons learned from establishing, maintaining, and authenticating the pyrethroid-resistant An. gambiae s.s. Muleba-Kis strain at the KCMUCo-PAMVERC Test Facility are described in this paper. Methods: Male mosquitoes from the F1 generation of wild-pyrethroid resistant mosquitoes were cross-bred with susceptible female An. gambiae s.s. Kisumu laboratory strain followed by larval selection using a pyrethroid insecticide solution. Periodic screening for phenotypic and genotypic resistance was done. WHO susceptibility tests and bottle bioassays were used to assess the phenotypic resistance, while Taqman™ assays were used to screen for known target-site resistance alleles (kdr and ace-1). Additionally, the strains were periodically assessed for quality control by monitoring adult weight and wing length. Results: By out-crossing the wild mosquitoes with an established lab strain, a successful resistant insectary colony was established. Intermittent selection pressure using alphacypermethrin has maintained high kdr mutation (leucine-serine) frequencies in the selected colony. There was consistency in the wing length and weight measurements from the year 2016 to 2020, with the exception that one out of four years was significantly different. Mean annual wing length varied between 0.0142–0.0028 mm compared to values obtained in 2016, except in 2019 where it varied by 0.0901 mm. Weight only varied by approximately 0.001 g across four years, except in 2017 where it differed by 0.005 g. Routine phenotypic characterization on Muleba-Kis against pyrethroids using the WHO susceptibility test indicated high susceptibility when type I pyrethroids were used compared to type II pyrethroids. Dynamics on susceptibility status also depended on the lapse time when the selection was last done. Conclusions: This study described the procedure for introducing, colonizing, and maintaining a resistant An. gambiae s.s. strain in the laboratory with leucine to serine substitution kdr allele which reflects the features of the wild-resistant population in East Africa. Challenges in colonizing a wild-resistant mosquito strain were overcome by out-crossing between mosquito strains of desired traits followed by intermittent insecticide selection at the larval stage to select for the resistant phenotype.