To improve the draft assembly of subterranean clover, the BNG Irys® system provided affordable, high‐throughput physical maps of high contiguity to validate the draft and extend existing scaffolds. Effective anchoring of sequence scaffolds to genetic linkage groups, coupled with the use of the BioNano system, resulted in a high‐confidence chromosome‐level genome assembly. Tissue‐specific transcript profiling with RNA‐Seq technology delivered gene expression data of high value for gene annotation of the assembly (Tsub_Refv2.0) and transcriptional dynamics to understand tissue‐specific pathways. This new genomic information is the key to identifying loci governing traits that allow marker‐assisted breeding in subterranean clover for comparative mapping with other species and the identification of tissue‐specific gene promoters for biotechnological improvement of forage legumes.