Abstract Current advances in next-generation sequencing (NGS) technology and assembling programs permit construct chromosome-level genome assemblies in various plants. In contrast to resequencing, the genome sequences provide comprehensive annotation data useful for plant genetics and breeding. Herein, we constructed a reference-based genome assembly of winter barley (H. vulgare ssp. vulgare) cv. ‘Hayakiso 2’ using long and short read NGS data and barley reference genome sequences from ‘Morex’. We constructed ‘Hayakiso 2’ genome sequences covering 4.3 Gbp with 55,477 genes. Comparative genomics revealed that 14,106 genes had orthologs to two barley data, wheat (A, B, and D homoeologs, respectively), and rice. From the gene ontology analysis, 2,494 orthologs against wheat and rice but not two barley contained agricultural important genes, such as ‘response to biotic and abiotic stress’ and ‘metabolic process’. Phylogenetic analysis using 76 pangenome data indicated that ‘Hayakiso 2’ was clustered into Japanese-type genomes with unique alleles. ‘Hayakiso 2’ genome sequences showed known genes related to flowering and facilitated barley breeding through the development of various markers related to agronomically important alleles such as tolerance to various types of biotic and abiotic stress. Therefore, ‘Hayakiso 2’ genome sequences will be used for the further barley breeding.