Abstract..Interspecific pairings between MAT-1 and MAT-2 strains of seven species of Ceratocystis from conifer hosts resulted in partial interfertility in many cases. Perithecia from such crosses typically produced few ascospores, which were often misshapen and intermixed with cellular debris, and a low ascospore germination percentage was seen. Intraspecific pairings of strains of opposite mating type generally produced many perithecia with many ascospores of a high germination percentage. Mycelial morphology, cycloheximide resistance and isozyme markers were used to confirm that many of the interspecific pairings produced viable hybrid progeny. Tester strains of C. pinicola, C. coerulescens, and C. laricicola frequently crossed with other species. Because MAT-2 strains of the Ceratocystis species studied are capable of selfing due to unidirectional mating type switching, MAT-2 tester strains that fruited poorly or were self-sterile had to be selected. In a few cases, these MAT-2 strains were induced to self by pairing with MAT-1 or MAT-2 strains of other species. Hybrids yiedling ascospores formed only in crosses of strains of opposite mating type, but many interspecific pairings of strains of the same or opposite mating type resulted in the production of sterile perithecia. Partial interfertility between species based on a low number of misshapen ascospores was a good indicator of biological species, and such delineations corresponded well with species delimited by morphological and physiological phenotypes.