BACKGROUND Dengue is endemic in Saudi Arabia especially in Jeddah, Makkah, Asir, and Jazan areas where pyrethroids are widely used to control the vector, Aedes aegypti. Resistance of Ae. aegypti to pyrethroid insecticides has been reported from most of these areas. OBJECTIVE The present study was carried out in Jazan region in south-west Saudi Arabia to explore the resistance status of Ae. aegypti to pyrethroids and the consequent underlying mechanisms. METHODS Three pyrethroids (permethrin, lambda-cyhalothrin, and cyfluthrin) were used to investigate the resistance status of Ae. aegypti adults following World Health Organization (WHO) standard methods: PCR and sequencing techniques were used to detect the S989P, V1016G and F1534C kdr mutations. RESULTS Ae. aegypti populations were susceptible to cyfluthrin and having a possibility of resistance to permethrin while resistant to lambda-cyhalothrin. Three potential kdr mutations were detected for the first time in Ae. aegypti population, F1534C, V106G, and S989P. It was found that F1534C often co-exists with V1016G and this haplotype was strongly associated with permethrin and lambda-cyhalothrin resistance. On the other hand, S989P mutation was detected as RR in 18.8% with a low-frequency rate (R) of 18.8%, and in 55.5% as R with 58.3% frequency rate in permethrin and lambda-cyhalothrin- resistant female mosquitoes, respectively. CONCLUSIONS Early detection of resistance alleles should be considered the essential tool for the successful implementation of insecticide resistance management strategies by providing early warning of insect resistance.