The prickly nightshadeSolanum rostratum, an annual malignant weed, is native to North America and has globally invaded 34 countries, causing serious threats to ecosystems, agriculture, animal husbandry, and human health. In this study, we constructed a chromosome-level genome assembly and annotation ofS. rostratum. The contig-level genome was initially assembled in 898.42 Mb with a contig N50 of 62.00 Mb from PacBio high-fidelity reads. With Hi-C sequencing data scaffolding, 96.80% of the initially assembled sequences were anchored and orientated onto 12 pseudo-chromosomes, generating a genome of 869.69 Mb with a contig N50 of 72.15 Mb. We identified 649.92 Mb (72.26%) of repetitive sequences and 3,588 non-coding RNAs in the genome. A total of 29,694 protein-coding genes were predicted, with 28,154 (94.81%) functionally annotated genes. We found 99.5% and 91.3% complete embryophyta_odb10 genes in the pseudo-chromosomes genome and predicted gene datasets by BUSCO assessment. The present genomic resource provides essential information for subsequent research on the mechanisms of environmental adaptation ofS. rostratumand host shift in Colorado potato beetles.