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Spodoptera frugiperdais a major agricultural pest that has invaded China since January 2019. Given that most of the individuals present in China carried the diagnostic rice-strain mtDNA (COI-RS), there was no efficient method to distinguish populations of S. frugiperda.In this study, we identified and characterized two variant microsatellite alleles in the mitochondrial NAD6gene of S. frugiperdaretrieved from the National Center for Biotechnology Center GenBank. We then sequenced partial NAD6genes containing the microsatellite region and the diagnostic COIbarcoding gene (used to distinguish the corn-strain and the rice-strain) of 429 invasive S. frugiperdaindividuals that were collected from the main infested regions in China during 2019–2020. Our data indicates that two kinds of interrupted repeat sequences, (ATA)4T(ATA)3and (ATA)5T(ATA)3, exist in the microsatellite region which we defined as the deletion type (NAD6-D), and the insertion type (NAD6-I) based on the repeat units’ differentiation, respectively. The presence of these two microsatellite types in the mtDNA genome of S. frugiperdawas further confirmed with the sequencing results in 429 samples. Moreover, NAD6-I and NAD6-D types were both present in individuals with COI-RS, while only NAD6-D type was detected in the COI-CS individuals. Interestingly, the two microsatellite types suggested a possible geographic distribution: the western migratory route (Yunan and Chongqing) was comprised exclusively of NAD6-I type, while both NAD6-I and NAD6-D types were identified in the predicted eastern migration trajectories (Hainan, Guangxi, Shandong, etc.). These results suggested that NAD6-D and NAD6-I types may be useful in distinguishing between populations, analyzing the evolutionary mechanism of mtDNA microsatellite polymorphism, inferring the migratory route of S. frugiperdain China, and developing precise and integrated control strategies for S. frugiperda.