Chloroplasts fuel plant development and growth by converting solar energy into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated mutant of barley ( ) has greatly broadened the field of chloroplast biology, including the discovery of retrograde signaling. Here, we report identification of the gene (also known as ) by positional cloning as well as its functional validation based on independently induced mutants by Targeting Induced Local Lesions in Genomes (TILLING) and RNA-guided clustered regularly interspaced short palindromic repeats-associated protein 9 endonuclease-mediated gene editing. The phenotypes of the independent mutants imply residual activity of HvCMF7 in the original allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. is a homolog of the Arabidopsis ( ) ( ) transcription factor gene , which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Notably, in barley we localized HvCMF7 to the chloroplast, without any clear evidence for nuclear localization.