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Abstract The cabbage looper,Trichoplusia ni, is a globally distributed highly polyphagous herbivore and an important agricultural pest.T. nihas evolved resistance to various chemical insecticides, and is one of the only two insect species that have evolved resistance to the biopesticideBacillus thuringiensis(Bt) in agricultural systems and has been selected for resistance to baculovirus infections. We report a 333‐Mb high‐qualityT. nigenome assembly, which has N50 lengths of scaffolds and contigs of 4.6 Mb and 140 Kb, respectively, and contains 14,384 protein‐coding genes. High‐density genetic maps were constructed to anchor 305 Mb (91.7%) of the assembly to 31 chromosomes. Comparative genomic analysis ofT. niwithBombyx morishowed enrichment of tandemly duplicated genes inT. niin families involved in detoxification and digestion, consistent with the broad host range ofT. ni. High levels of genome synteny were found betweenT. niand other sequenced lepidopterans. However, genome synteny analysis ofT. niand theT. niderived cell line High Five (Hi5) indicated extensive genome rearrangements in the cell line. These results provided the first genomic evidence revealing the high instability of chromosomes in lepidopteran cell lines known from karyotypic observations. The high‐qualityT. nigenome sequence provides a valuable resource for research in a broad range of areas including fundamental insect biology, insect‐plant interactions and co‐evolution, mechanisms and evolution of insect resistance to chemical and biological pesticides, and technology development for insect pest management.