Precise kinetochore-microtubule interactions ensure faithful chromosome segregation in eukaryotes. Centromeres, identified as scaffolding sites for kinetochore assembly, are among the most rapidly evolving chromosomal loci in terms of the DNA sequence and length and organization of intrinsic elements. Neither the centromere structure nor the kinetochore dynamics is well studied in plant-pathogenic fungi. Here, we sought to understand the process of chromosome segregation in the rice blast fungus Magnaporthe oryzae High-resolution imaging of green fluorescent protein (GFP)-tagged inner kinetochore proteins CenpA and CenpC revealed unusual albeit transient declustering of centromeres just before anaphase separation of chromosomes in M. oryzae Strikingly, the declustered centromeres positioned randomly at the spindle midzone without an apparent metaphase plate per se Using CenpA chromatin immunoprecipitation followed by deep sequencing, all seven centromeres in M. oryzae were found to be regional, spanning 57-kb to 109-kb transcriptionally poor regions. Highly AT-rich and heavily methylated DNA sequences were the only common defining features of all the centromeres in rice blast. Lack of centromere-specific DNA sequence motifs or repetitive elements suggests an epigenetic specification of centromere function in M. oryzae PacBio genome assemblies and synteny analyses facilitated comparison of the centromeric/pericentromeric regions in distinct isolates of rice blast and wheat blast and in Magnaporthiopsis poae Overall, this study revealed unusual centromere dynamics and precisely identified the centromere loci in the top model fungal pathogens that belong to Magnaporthales and cause severe losses in the global production of food crops and turf grasses.IMPORTANCEMagnaporthe oryzae is an important fungal pathogen that causes a loss of 10% to 30% of the annual rice crop due to the devastating blast disease. In most organisms, kinetochores are clustered together or arranged at the metaphase plate to facilitate synchronized anaphase separation of sister chromatids in mitosis. In this study, we showed that the initially clustered kinetochores separate and position randomly prior to anaphase in M. oryzae Centromeres in M. oryzae occupy large genomic regions and form on AT-rich DNA without any common sequence motifs. Overall, this study identified atypical kinetochore dynamics and mapped functional centromeres in M. oryzae to define the roles of centromeric and pericentric boundaries in kinetochore assembly on epigenetically specified centromere loci. This study should pave the way for further understanding of the contribution of heterochromatin in genome stability and virulence of the blast fungus and its related species of high economic importance.Copyright © 2019 Yadav et al.