The SQUAMOSA-promoter binding like (SPL) gene family encodes transcription factors that have been shown in many species to influence plant growth and development, but information about these genes in barley (Hordeum vulgare L.) is limited. This study identified 17 barley SPL genes, within eight distinct groups, that are orthologs of SPL genes described in Arabidopsis, wheat, and rice. Sixteen barley SPLs undergo alternative splicing. Seven SPLs contain a putative miR156 target site and the transcript levels of the miR156-targeted HvSPLs (HvSPL3, 13 and 23) were lower in vegetative than in reproductive phase but this was true also for some SPL genes such as HvSPL6 that were not regulated by miR156. Because SPL gene products regulate miR172, which is also involved in floral development, the expression of miR172 was studied. An antagonistic expression pattern of miR156 and miR172b during the vegetative and the reproductive phases signifies their apparent function in barley growth phase transition. Characterization of a barley mir172 mutant having an abnormal, indeterminate spikelet phenotype suggests the possible feedback role of AP2/miR172 module on HvSPL genes. This is the first comprehensive analysis of the miR156/SPL/miR172 axis in barley that provides a basis to elucidate their roles in various biological processes.