The yellow fever mosquito, Aedes aegypti L., known for transmitting viruses causing yellow fever, dengue, chikungunya, and Zika fever, presents a substantial risk to global human health. The development of insecticide resistance in disease vectors has become a significant problem in Ae. aegypti. Monitoring insecticide resistance is essential for resistance management in Ae. aegypti. This study involved the collection of Ae. aegypti populations from four important cotton-growing regions in southern Punjab, Pakistan, for resistance monitoring over a two-year period (2021-2022). This study also assessed the impact of insecticide resistance on biological parameters of Ae. aegypti. Moderate-to-high levels of resistance were observed against all the tested insecticides viz., chlorpyrifos, chlorfenapyr, deltamethrin, flonicamid, spirotetramat, and spinetoram. However, compared to the Lab-susceptible population, higher levels of resistance to buprofezin (59.03-84.40) and imidacloprid (68.49-100.01) were found in all populations. This high resistance can be attributed to increased use of these two insecticides in cotton fields, as compared to other insecticides. In the lab-susceptible population, higher values for the intrinsic rate of increase (r) and the net reproductive rate (R