BACKGROUND Pyrethroid resistance is a threat to effective vector control of Aedes aegypti, the vector of dengue, Zika and other arboviruses, but there are many major knowledge gaps on the mechanisms of resistance. In Jeddah and Makkah, the principal dengue-endemic areas of Saudi Arabia, pyrethroids are used widely for Ae. aegypti control but information about resistance remains sparse, and the underlying genetic basis is unknown. Findings from an ongoing study in this internationally significant area are reported here. METHODS Aedes aegypti collected from each city were raised to adults and assayed for resistance to permethrin, deltamethrin (with and without the synergist piperonyl butoxide, PBO), fenitrothion, and bendiocarb. Two fragments of the voltage-gated sodium channel (Vgsc), encompassing four previously identified mutation sites, were sequenced and subsequently genotyped to determine associations with resistance. Expression of five candidate genes (CYP9J10, CYP9J28, CYP9J32, CYP9M6, ABCB4) previously associated with pyrethroid resistance was compared between assay survivors and controls. RESULTS Jeddah and Makkah populations exhibited resistance to multiple insecticides and a similarly high prevalence of resistance to deltamethrin compared to a resistant Cayman strain, with a significant influence of age and exposure duration on survival. PBO pre-exposure increased pyrethroid mortality significantly in the Jeddah, but not the Makkah strain. Three potentially interacting Vgsc mutations were detected: V1016G and S989P were in perfect linkage disequilibrium in each strain and strongly predicted survival, especially in the Makkah strain, but were in negative linkage disequilibrium with 1534C, though some females with the Vgsc triple mutation were detected. The candidate gene CYP9J28 was significantly over-expressed in Jeddah compared to two susceptible reference strains, but none of the candidate genes was consistently up-regulated to a significant level in the Makkah strain. CONCLUSIONS Despite their proximity, Makkah and Jeddah exhibit significant differences in pyrethroid resistance phenotypes, with some evidence to suggest a different balance of mechanisms, for example with more impact associated with CYP450s in the Jeddah strain, and the dual kdr mutations 989P and 1016G in the more resistant Makkah strain. The results overall demonstrate a major role for paired target site mutations in pyrethroid resistance and highlight their utility for diagnostic monitoring.