A row-type mutant of barley named poly-row-and-branched spike (prbs) was previously obtained from a two-rowed cultivar Pudamai-2 after treated by inflorescence soaking in maize total DNA solution. The mutant produces branched spikes with irregular multiple rows. Genetic analysis indicated that the mutant phenotype was caused by a recessive gene prbs, and the PRBS locus had a recessive epistatic effect on an independent locus (denoted as Vrsx) conferring the variation of two-rowed spike vs. six-rowed spike. This study aimed to map PRBS as well as Vrsx using simple sequence repeats (SSR) markers. We developed an F2 population from a cross between the prbs mutant and a six-rowed cultivar Putianwudu for the gene mapping. As the two target loci interacted to result in a segregation ratio of two-rowed type:six-rowed type:prbs=9:3:4 in the population, we adopted a special strategy to map the two loci. PRBS was mapped between SSR markers HvLTPPB and Bmag0508A on the short arm of chromosome 3H, with distances of 24.7 and 14.3 cM to the two markers, respectively. Vrsx was mapped between SSR markers Bmag0125 and Bmag0378 on chromosome 2H, with distances of 6.9 and 15.3 cM to the two markers, respectively. This suggests that Vrsx should be the known locus Vrs1, which predominantly controls row-type variation in barley cultivars, and PRBS is a new locus related to the row type of spikes in barley. A row-type mutant of barley named poly-row-and-branched spike (prbs) was previously obtained from a two-rowed cultivar Pudamai-2 after treated by inflorescence soaking in maize total DNA solution. rows. Genetic analysis indicated that the mutant phenotype was caused by a recessive gene prbs, and the PRBS locus had a recessive epistatic effect on an independent locus (denoted as Vrsx) conferring the variation of two-rowed spike vs. six-rowed spike. We developed an F2 population from a cross between the prbs mutant and a six-rowed cultivar Putianwudu for the gene mapping. As the two target loci interacted to result in a segregation ratio of two-rowed type: six-rowed type: prbs = 9: 3: 4 in the population, we adopted a special strategy to map the two loci. PRBS was mapped between SSR markers HvLTPPB and Bmag0508A on the short arm of chromosome 3H, with distances of 24.7 and 14.3 cM to the two markers, respectively. Vrsx was mapped between SSR markers Bmag0125 and Bmag0378 on chromosome 2H, with distances of 6.9 and 15.3 cM to the two markers, respectively. This suggests that Vrsx should be known locus Vrs1, which predominantly controls row-type variation in barley cultivars, and PRBS is a new locus related to the row type of spikes in barley.