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Ceratocystis sensu stricto is a genus of closely related fungi that are mostly plant pathogens. Morphological variation between species in this genus is limited, and species delineation is strongly reliant on phylogenetic inference. Primary support for many of the species is based on the ITS region which, on its own, can be used to delineate all species described in the genus. However, the recent discovery of two ITS types in a single isolate of Ceratocystis questions the use of this marker in taxonomic studies. The aim of this study was to consider the potential use of alternative gene regions to support the species boundaries in this genus. The phylogenetic value of the βT 1 and EF 1-α gene regions, generally used in combination with ITS, were re-evaluated and compared to five single-copy protein coding genes (CAL, RPBII, MS204, FG1093 and Mcm7). As an alternative approach, genome-wide …