Early cytological studies indicated that chromatids of mitotic chromosomes coil into a helix but a direct proof for this assumption was missing. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy to reveal a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingled due to the stochastic positioning of chromatin loops. Helical turn size varied along chromosome length, positively correlating with chromatin and negatively with gene density. Comparison with previously published animal studies suggested minor differences in higher order chromatin organisation between plants and animals.