De novo centromeres refer to ectopic centromeres that acquire functionality in genomic regions lacking native centromeric repeat sequences. To investigate mechanisms underlying de novo centromere formation, we employed a multi-omics approach integrating genomic, transcriptomic, and epigenetic data from wheat–barley disomic addition line 7HS**. The barley 7HS chromosome arm forms a 4.7 Mbp de novo centromere without canonical repeats, revealed by specific CENH3 enrichment. The emergence of de novo centromere is characterized by reduced repetitive DNA content and earlier LTR retrotransposon insertions compared to native centromeres. Notably,we identified a GT-rich microsatellite,designated CentHv7,within this region—a sequence also conserved in the native centromeres of barley. We found that R-loops,three-stranded nucleic acid complexes,are preferentially enriched in the barley centromeric regions. In the de novo centromeric region of 7HS**,a de novo R-loop forms at exons 11 and 12 of the upregulated gene HORVU.MOREX.r3.7HG0694980,co-localizing with bivalent chromatin modifications H3K4me3 and H3K27me3. In conclusion,our findings emphasize that the sequence features of de novo centromeres,in combination with unique gene expression patterns,newly formed R-loop structures,and specialized poised chromatin states,may collectively create a unique epigenetic environment that correlates with the formation and stabilization of de novo centromeres in barley.