AbstractPlantagois a major genus belonging to the Plantaginaceae family and is used in herbal medicine, functional food, and pastures. SeveralPlantagospecies are also characterized by their global distribution, but the mechanism underpinning this is not known. Here, we present a high-quality, chromosome-level genome assembly ofPlantago majorL., a species ofPlantago, by incorporating Oxford Nanopore sequencing and Hi-C technologies. The genome assembly size was approximately 671.27 Mb with a contig N50 length of 31.30 Mb. 31,654 protein-coding genes were identified from the genome. Evolutionary analysis showed thatP. majordiverged from other Lamiales species at ~62.18 Mya and experienced two rounds of WGD events. Notably, many gene families related to plant acclimation and adaptation expanded. We also found that many polyphenol biosynthesis genes showed high expression patterns in roots. Some amino acid biosynthesis genes, such as those involved in histidine synthesis, were highly induced under metal (Ni) stress that led to the accumulation of corresponding metabolites. These results suggest persuasive arguments for the global distribution ofP. majorthrough multiscale analysis. Decoding theP. majorgenome provides a valuable genomic resource for research on dissecting biological function, molecular evolution, taxonomy, and breeding.