[Aim] Recent research has found that Spodoptera frugiperda, Spodoptera exigua, Spodoptera litura, and Mythimna separata often occur simultaneously in fields. Because of similar morphological characteristics of adults and larvae, it is difficult to differentiate S. frugiperda from the other three species using traditional morphological identification methods. It is necessary to develop a rapid method to differentiate S. frugiperda from the other morphologically similar species.[Method] This study analyzed the restriction enzyme recognition sites in the mtCOI (mitochondrial cytochrome oxidase subunit I) of S. frugiperda, S. exigua, S. litura, and M. separata. A new forward primer of the target fragment of mtCOI gene was designed, which was used to amplify the targeted production.[Result] In the 556-561 bp of mtCOI segment, Sbf I enzyme recognition site was found in all individuals of S. frugiperda, while there was no Sbf I restriction enzyme recognition sites in S. liitura, S. exigua, and M. separata. The results showed that the PCR products of S. frugiperda could be digested by Sbf I, and a characteristic band of 420 bp was found. In contrast, the PCR products of S. litura, S. exigua, and M. separata could not be digested by Sbf I.[Conclusion] The mtCOI PCR-RFLP technique developed in this study can differentiate S. frugiperda from the other three species effectively, which provides a rapid method for the identification of S. frugiperda.