Proteins play a major role in the 3D organization of nuclear genome and its function. While histones arrange DNA into nucleosomes, other proteins contribute to higher-order chromatin structures. We purified barley mitotic metaphase chromosomes and identified their proteins. This included proteins that were not known to be present on chromosomes. The experimentally validated presence of a nucleolar protein FIBRILLARIN 1 on metaphase chromosomes, suggested that plant chromosomes are coated by a perichromosomal protein layer during mitosis. These results improve the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.