Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. How centromeres form in strongly host-adapted fungal pathogens has yet to be investigated. Here, we characterized the centromere structures in closely related species of mammalian-specific pathogens of the fungal phylum of Ascomycota. Methods allowing reliable continuous culture ofPneumocystisspecies do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that thePneumocystisCENP-A ortholog is functionally equivalent to CENP-ACnp1ofSchizosaccharomyces pombe. Using organisms from a short-termin vitroculture or infected animal models and ChIP-seq, we identified centromeres in threePneumocystisspecies that diverged ~100 million years ago. Each species has a unique short regional centromere (< 10kb) flanked by heterochromatin in 16–17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. CENP-C, a scaffold protein that links the inner centromere to the kinetochore appears dispensable in one species, suggesting a kinetochore rewiring. Despite the loss of DNA methyltransferases, 5-methylcytosine DNA methylation occurs in these species, though not related to centromere function. These features suggest an epigenetic specification of centromere function.