R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric ( chromatin in wild-type budding yeast. Here we used an strain that accumulates R-loops to investigate the consequences of R-loops at chromatin and chromosome segregation. We show that Hpr1 interacts with the -histone H3 variant, Cse4, and prevents the accumulation of R-loops at chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at chromatin that was reduced by overexpression of in strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at chromatin were observed in strains. We determined that accumulation of R-loops at chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by overexpression in strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at contributes to defects in kinetochore integrity and CIN.