The purpose of this study was to determine the sensitivity of a field population of Spodoptera frugiperda (Lepidoptera: Noctuidae) in China to six insecticides, detect the genotype and mutation frequency of the Acetylcholine esterase (AChE) gene Ace-1, a target of insecticides, and inform the scientific use of insecticides in the field. Larvae of S. frugiperda were collected from maize fields in Anhui, Hunan, Guizhou, Guangdong, Hainan, Guangxi, and Yunnan. The sensitivity to emamectin benzoate, pyridalyl, chlorantraniliprole, flubendiamide, indoxacarb, and lambda-cyhalothrin was determined by the leaf dipping method, and the total DNA of 80 individuals from 7 populations was extracted. The genotypes of Ace-1 and the mutation frequencies of the target mutation sites were determined by sequence alignment and sequencing peak analysis. The results of the bioassay demonstrated that larvae from the seven field populations were sensitive to emamectin benzoate, pyridalyl, chlorantraniliprole, flubendiamide, indoxacarb, and lambda-cyhalothrin. The molecular detection results showed that there were heterozygous mutations in the Ace-1 gene at the A201S and G227A loci. In conclusion, the seven field populations of S. frugiperda were sensitive to emamectin benzoate, pyridalyl, chlorantraniliprole, flubendiamide, indoxacarb, and lambda-cyhalothrin; however, these seven field populations all carry resistance genes to organophosphorus and carbamate insecticides. Therefore, our results indicate a potential risk for S. frugiperda resistance and the resistance monitoring should be strengthened.