In the city of Magelang, Indonesia, the distribution of Dengue Haemorhagic Fever (DHF) cases tend to be clustered, ever changing along with human urbanization from 2014 to 2017. Although DHF cases have been less reported in the city of Magelang for the past 5 years, vector control measures by using insecticide space spraying, particularly permethrin, have been continuously performed. Current study aimed to detect kdr mutations associated with pyrethroid resistance in Ae. aegypti and to study possible association between insecticide resistance and DHF case distribution related to human urbanization. The study was a cross sectional study conducted in 3 sub-districts in the city of Magelang, Central Java, Indonesia. Eggs of Ae. aegypti collected from 195 sample households were reared and were tested for resistance to pyrethroids by using PCR. Primers AaSCF1 and AaSCR4, and primers AaSCF7 and AaSCR7 were used in detecting presence of mutation in VGSC IIS6 and IIIS6 gene, respectively. Fragments of amplified DNA were sequenced and were analyzed. Spatio-temporal using Standard Deviational Ellipse (SDE) was performed to obtain mapping of DHF case distribution trends. The total number of DHF case was 380 cases, with the most cases (158) occurred in 2015 and the least cases (66) reported in 2017. DHF case distribution was grouped into several clusters. SDE calculation demonstrated movement of DHF case in the direction to principal arterial road, suggesting link to urbanization. Gene sequencing demonstrated VGSC IIS6 gene mutation (S989P and V1016G) in Ae. aegypti collected from study areas, indicating resistance to permethrin. VGSC IIIS6 gene mutation was not found. Current study concluded that multiple kdr mutations associated with resistance to pyrethroid was detected in Ae. aegypti, and that human urbanization may have a role in the development of such resistance.