To determine the feasibility of using internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal DNA sequences as genetic markers in molecular systematic and phylogenetic studies of Ophiostoma, the presence of multiple types of ITS in the two sibling sapstain fungi, Ophiostoma piceae and O. quercus were examined in 22 isolates from diverse geographic regions. PCR and sequence analysis revealed that two different versions of ITS sequences were present in the PCR-products amplified with the ITS primers, ITS1-ITS4 or ITS5-ITS4 or ITS1-F-ITS4, in every isolate of the two Ophiostoma species tested. Surprisingly, the two ITS sequences were shared by both species. One ITS sequence which was major in O. piceae was designated as the OPC type, and the other ITS sequence which was minor in O. piceae was designated as the OPH type. In O. quercus the OPH type was major and the OPC type was minor. The major ITS comprised more than 99% of the amplicons in both species. The results suggest that common ITS gene pools have been maintained at different levels within these sibling species.