Chrysoporthe cubensis is one of the most important pathogens of Eucalyptus. Based on phylogenetic evidence and geographic origin, isolates of this fungus are known to reside in distinct ‘South America’ and ‘Southeast Asia’ clades. In this study, reproductive isolation amongst these isolates of C. cubensis was tested using gene flow statistics for 12 polymorphic loci, and to support these data, phylogenetic affiliations based on gene trees and a multigene phylogeny were used. Gene flow statistics between populations, and relative to the closely related Chrysoporthe austroafricana, were low and not significantly different (P < 0.05). Additionally, phylogenetic analyses of DNA sequence data for four gene regions convincingly distinguished the two subclades of C. cubensis. Isolates in the Southeast Asian subclade are described in the new species, Chrysoporthe deuterocubensis. Chrysoporthe cubensis and C. deuterocubensis represent closely related fungi that are thought to be native to South America and Southeast Asia, respectively. A technique is presented that allows for rapid differentiation between these species and that will aid in quarantine procedures to limit their spread to new environments.