Abstract: The fall armyworm (FAW), Spodoptera frugiperda, is a significant pest of many crops in the world and it is native to the Western Hemisphere, where the species has shown the ability to rapidly evolve resistance to transgenic plants and insecticides. Despite the importance of this species, there is a gap in the knowledge regarding the migration pattern of FAW in South America. Here, we examined the genetic diversity of FAW populations across a wide agricultural area of Brazil and three locations from Argentina using a Genotyping-by-Sequencing (GBS) approach. We also characterized samples by their host strain based on mitochondrial and Z-linked genetic markers. The GBS methodology enabled us to discover 3309 SNPs, including neutral and outlier markers. The 37 mitochondrial haplotypes did not reveal clusters based on geography, whereas GBS data revealed significant genetic structure between Brazil and Argentina, and also among the Argentinian ecoregions. Populations inside Brazil showed little genetic differentiation indicating high gene flow among locations and confirming that structure is related to the presence of corn and rice strains. Outlier analysis indicated 456 loci putatively under selection, including genes possibly related to resistance evolution. This study provides clarification of the population genetic structure of FAW in South America and highlights the importance of genomic research to understand the risks of spread of resistance genes Ver menos