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.