Cotton aphid, Aphis gossypii Glover, 1877 (Hemiptera: Aphididae), is a major pest in cotton fields. Neonicotinoids are important and highly prevalent insecticides currently used against A. gossypii and other herbivorous insect pests in the Mediterranean Region of Turkey. However, some insecticide applications against A. gossypii in the Cukurova Region have failed despite using high rates. Therefore, bioassays and enzyme analyses were conducted to determine resistance to imidacloprid and thiamethoxam in A. gossypii populations collected in 2015-2016 from cotton fields in this region. Resistance factors (RF) were 54.6 to 206.5 fold for imidacloprid and 5.7 to 65.7 fold for thiamethoxam. Populations from Kurkculer (RF 206.5) had the highest LD50 for imidacloprid and from Korkuyu (RF 65.7) for thiamethoxam. Enzyme analysis revealed statistically higher metabolic resistance. Maximum enzyme activities were 17.8, 142.3 and 3.8 nM/min/mg protein for carboxylesterase for in Korkuyu, for glutathione S-transferase in Bahce and for cytochrome P450 monooxygenase in Korkuyu, respectively. This study revealed the development of resistance in A. gossypii to neonicotinoid insecticides in Turkey and the need for new management strategies to break this resistance.