Abstract: Development of a novel method for identifying centromeric DNA sequences ChIP using anti-CENH3 antibodies is a powerful tool for isolation of centromeric DNAs.However, ChIP requires high quality antibodies, and it is sometimes difficult to raise high quality antibodies, which is an obstacle for the isolation.Here, we report a novel method for the isolation, based on the fact that the sizes between canonical and centromeric nucleosomes are different.In maize and rice, shorter DNA fragments on the CENH3 nucleosomes than those on the canonical H3 nucleosomes have been reported.To know its universality, we isolated chromatins from rice, tobacco and welsh onion, and digested them into mononucleosome sizes.The digested DNA was separated using agarose gels.Monomeric size and shorter DNA fragments were purified, and used as templates of qPCR.In the qPCR, centromeric DNAs were significantly concentrated in the shorter fractions.In addition, we found the shorter fragments enrichment with the centromeric DNA sequences by sequence-length comparison after Illumina sequencing.These results imply shorter DNA fragments on centromeric nucleosomes are a universal feature, which must be useful for identifying centromeric DNAs without anti-CENH3 antibodies.