Hybridization is recognized as a notable driver of evolution and adaptation, which closely related species may exploit in the form of incomplete reproductive barriers. Three closely related species ofCeratocystis(i.e.,C. fimbriata,C. manginecansandC. eucalypticola) have previously been shown to hybridize. In such studies, naturally occurring self-sterile strains were mated with an unusual laboratory-generated sterile isolate type, which could have impacted conclusions regarding the prevalence of hybridization and inheritance of mitochondria. In the current study, we investigated whether interspecific crosses between fertile isolates of these three species are possible and, if so, how mitochondria are inherited by the progeny. For this purpose, a PCR-RFLP method and a mitochondrial DNA-specific PCR technique were custom-made. These were applied in a novel approach of typing complete ascospore drops collected from the fruiting bodies in each cross to distinguish between self-fertilizations and potential hybridization. These markers showed hybridization betweenC. fimbriataandC. eucalypticolaand betweenC. fimbriataandC. manginecans, while no hybridization was detected in the crosses involvingC. manginecansandC. eucalypticola. In both sets of hybrid progeny, we detected biparental inheritance of mitochondria. This study was the first to successfully produce hybrids from a cross involving self-fertile isolates ofCeratocystisand also provided the first direct evidence of biparental mitochondrial inheritance in theCeratocystidaceae. This work lays the foundation for further research focused on investigating the role of hybridization in the speciation ofCeratocystisspecies and if mitochondrial conflict could have influenced the process.