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Abstract In order to genetic analysis of spike characteristics in barley, an experiment was conducted with 100 F3 and F4 barley families derived from Badia × Komino cross at Research Farm college of Agricultural University of Gonbad Kavous (Iran) based on randomized complete block design with three replications. Agronomic traits such as spike length, number of seeds per spike, total of spike, total weight of spike, grain length and grain diameter were measured. Linkage map with 7 SSR and 69 polymorphic alleles of iPBS markers were prepared which covered 632.2 cM of barley genome. QTL analysis was performed based on the method of composite interval mapping (CIM). Ten QTLs (with additive effect ranged from 127.07 for spike number to -0.625 mm for grain length) were detected. Phenotypic variance explained by QTLs ranged from 10.9 to 12.9 percent, which the highest related to spike length in F3 generation and the lowest related to the total number of spikes in F3 generation and the total weight of spike in F4 generation. All detected QTL were major effects and after validation can be used in breeding programs and marker-assisted selection.