Six-rowed spike 1 (Vrs1) is a gene of major importance for barley breeding and germplasm management as it is the main gene determining spike row-type (2-rowed vs. 6-rowed). This is a widely used DUS trait, and has been often associated to phenotypic traits beyond spike type. Comprehensive re-sequencing Vrs1 revealed three two-rowed alleles (Vrs1.b2; Vrs1.b3; Vrs1.t1) and four six-rowed (vrs1.a1; vrs1.a2; vrs1.a3; vrs1.a4) in the natural population. However, the current knowledge about Vrs1 alleles and its distribution among Spanish barley subpopulations is still underexploited. We analyzed the gene in a panel of 215 genotypes, made of Spanish landraces and European cultivars. Among 143 six-rowed accessions, 57 had the vrs1.a1 allele, 83 were vrs1.a2, and three showed the vrs1.a3 allele. Vrs1.b3 was found in most two-rowed accessions, and a new allele was observed in 7 out of 50 two-rowed Spanish landraces. This allele, named Vrs1.b5, contains a ‘T’ insertion in exon 2, originally proposed as the causal mutation giving rise to the six-row vrs1.a2 allele, but has an additional upstream deletion that results in the change of 15 amino acids and a potentially functional protein. We conclude that eight Vrs1 alleles (Vrs1.b2, Vrs1.b3, Vrs1.b5, Vrs1.t1, vrs1.a1, vrs1.a2, vrs1.a3, vrs1.a4) discriminate two and six-rowed barleys. The markers described will be useful for DUS identification, plant breeders, and other crop scientists.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness grants AGL2010-21929, AGL2013-48756-R, RFP2012-00015-00-00, RTA2012-00033-C03-02, and EUI2009-04075 (national code for Plant-KBBE project ExpResBar). CPC was funded by the Spanish Ministry of Economy, Industry and Competitiveness grant no. BES-2011-045905 (linked to project AGL2010-21929). TK and SS were supported by a research fund by the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation grants no. TRS1002). SS was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellow for Research Abroad and a Grant-in-Aid for Young Scientists (B) (no. 16 K18635).