The organization of centromeric chromatin of diploid barley (Hordeum vulgare) encoding two (α and β) CENH3 variants was analysed by super-resolution microscopy. Antibody staining revealed that both CENH3 variants are organized in distinct but intermingled subdomains in interphase, mitotic and meiotic centromeres. Artificially extended chromatin fibres illustrate that these subdomains are formed by polynucleosome clusters. Thus, a CENH3 variant-specific loading followed by the arrangement into specific intermingling subdomains forming the centromere region appears. The CENH3 composition and transcription vary among different tissues. In young embryos, most interphase centromeres are composed of both CENH3 variants, while in meristematic root cells, a high number of nuclei contain βCENH3 mainly dispersed within the nucleoplasm. A similar distribution and no preferential arrangement of the two CENH3 variants in relationship to the spindle poles suggest that both homologs meet the same function in metaphase cells.