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In small-grain cereals, grain yield is closely associated with grain number. Improved spikelet survival is an important trait for increasing grain yield. We investigated spikelet number, spikelet survival and yield-related traits under greenhouse conditions, and pot- and soil-grown field conditions. Thirty-two spring barley (Hordeum vulgare L.) accessions (14 two- and 18 six-rowed accessions) were manually dissected to determine spikelet/floret number on the main culm spike (SNS) at awn primordium (AP), tipping (TIP), heading and anther extrusion. We observed a significant difference between two- and six-rowed barley for SNS and spikelet survival at all stages and growing conditions. Both traits were highly genetically controlled, with repeatability and broad-sense heritability values of 0.74–0.93. The rate of spikelet survival from AP to harvest was higher in two- (~70%) than in six-rowed (~58%) barley. Spikelet …