The barley genome encodes a complete set of MADS-box proteins sharing homology with components of the ABCDE model, which explains the molecular basis of floral organ identity in angiosperm flowers. Although the E-class members are universally expressed across floral whorls and crucial for flower development in Arabidopsis and rice, the functional role of the barley E-class LOFSEP subfamily (comprising MADS1, MADS5, and MADS34) remains elusive, particularly during spikelet formation. Here, we characterize the single, double and triple lofsep mutants in barley in an attempt to overcome the anticipated genetic redundancy. Surprisingly, loss of function of all LOFSEP members only disturbs lemma development, either converting this hull organ into a leaf-like structure or reducing its size. The inner organs, including lodicules, anthers and pistil remain unaffected. A systematic interrogation of how ABCDE class genes are affected in all whorls of the mutants was undertaken. Generally, in the lemma and palea of the lofsep mutants, A- and E-class genes are hyperactivated, B- and C- classes are slightly repressed, and D-class genes show unchanged expression in these inner organs. Intriguingly, loss of function of MADS6, an AGL6 member closely related to the E-class genes, leads to most organs being transformed into lemma-like organs with new spikelets generated from the center of the flower. Contrasting with rice, these findings suggest barley LOFSEPs may have regressed in determining floral organ identity, and this could be partially compensated by HvMADS6.