The melon and cotton aphid Aphis gossypii Glover (Hemiptera; Aphididae) is one of the most serious pests worldwide. We surveyed insecticide susceptibility in A. gossypii field populations to 12 insecticides (6 neonicotinoids, 3 pyrethroids and 3 others) to examine resistance ratios. The levels of insecticide resistance were extremely high, especially to neonicotinoids, such as acetamiprid, clothianidin, thiacloprid and imidacloprid. To identify the neonicotinoid resistance mechanisms, we used an imidacloprid-resistant (IMI-R) strain as a model strain. IMI-R showed an extremely high resistance ratio and also cross-resistance to all the test neonicotinoids. However, there was little or no cross-resistance to the other insecticides, including sulfoxaflor. Synergist tests and enzyme activity assays suggested the absence of resistance mechanisms based on enhanced detoxification enzymes, such as cytochrome P450, esterase and glutathione S-transferase. One point mutation was found in the beta1 subunit loop D region of the nicotinic acetylcholine receptor (nAChR) of the IMI-R strain. This R81T point mutation was also found in field populations collected from 5 regions. Therefore, the R81T point mutation was identified as an important mechanism of imidacloprid resistance in A. gossypii.