The rapid wide-scale spread of fall armyworm (Spodoptera frugiperda) has caused serious crop losses34 globally. However, differences in the genetic background of subpopulations and the mechanisms of rapid adaptation behind the invasion are still not well understood. Here we report the assembly of a 390.38-M chromosome-level genome of fall armyworm using Pacific Bioscience (PacBio) and Hi-C sequencing technologies with scaffold N50 of 12.7 M consisting of 22260 annotated protein-coding genes. Genome-wide resequencing of 103 samples from 16 provinces in China revealed that the fall armyworm population comprises a complex inter-strain hybrid, mainly with the corn-strain genetic background and less of the rice-strain, which highlights the inaccuracy of strain identification using mitochondrial or Triosephosphate isomerase (Tpi) genes. Analysis of genes related to pesticide- and Bt-resistance showed that the risk of fall armyworm developing resistance to conventional pesticides is very high. Laboratory bioassay results showed that insects invading China carry resistance to organophosphate and pyrethroid pesticides, but are sensitive to genetically modified maize expressing Bacillus thuringiensis (Bt) toxins Cry1Ab in field experiments. Additionally, two mitochondrial fragments are inserted into the nuclear genome, and the insertion event occurred after the differentiation of the two strains. This study represents a valuable advance toward improving management strategies for fall armyworm.