Abstract: Centromere H3 proteins (CenH3s) are variants of histone H3 specialized for packaging centromere DNA. Unlike canonical H3 which is among the most conserved of eukaryotic proteins, CenH3s are rapidly evolving, raising questions about orthology and conservation of function across species. To gain insight on CenH3 evolution and function, a phylogenetic analysis was undertaken on CenH3 proteins drawn from a single, ancient lineage, the Fungi. Using maximum likelihood methods, a credible phylogeny was derived for the conserved histone fold domain (HFD) of twenty-five fungal CenH3s. The collection consisted mostly of hemiascomycetous yeasts, but also included basidiomycetes, euascomycetes, and an archaeascomycete. The HFD phylogeny closely recapitulated known evolutionary relationships between the species, supporting CenH3 orthology. The fungal CenH3s lacked significant homology in their N termini except for those of the Saccharomyces/Kluyveromyces clade which all contained a region homologous to the essential N-terminal domain found in Saccharomyces cerevisiae Cse4. The ability of several heterologous CenH3s to function in S. cerevisiae was tested and found to correlate with evolutionary distance. Domain-swapping between S. cerevisiae Cse4 and the noncomplementing Pichia angusta ortholog showed that species-specificity could not be explained by the presence or absence of any recognized secondary structural element of the HFD.