In Japan, wasabi (Eutrema japonicum) is an important traditional condiment, and is recognized as an endemic species. In the present study, we generated a chromosome-level and haplotype-resolved reference genome forE. japonicumusing PacBio CLR (continuous long reads), Illumina, and Hi-C sequencing data. The genome consists of 28 chromosomes that contain 1,512.1 Mb of sequence data, with a scaffold N50 length of 55.67 Mb. We also reported the subgenome and haplotype assignment of the 28 chromosomes by read-mapping and phylogenic analysis. Three validation methods (Benchmarking Universal Single-Copy Orthologs, Merqury, and Inspector) indicated that our obtained genome sequences were a high-quality and high-completeness genome assembly. Comparison of genome assemblies from previously published genomes showed that our obtained genome was of higher quality. Therefore, our genome will serve as a valuable genetic resource for both chemical ecology and evolution research of the generaEutremaand Brassicaceae, as well as for wasabi breeding.