Variation among isolates of the Sirococcus shoot blight pathogen (attributed to Sirococcus conigenus, Sirococcus sp., or one of many synonyms) was studied. Inter-simple-sequence-repeat-anchored polymerase chain reaction fingerprints, nuclear rDNA internal transcribed spacers and 5.8S sequences, and 18S rDNA sequences were analysed. In addition, culture morphology as well as sizes and shapes of conidia produced on seed cone scales in culture were compared. Two main groups were distinguished based on both genetic markers and cultural characteristics. The P group consists of isolates mostly from pine and spruce from Europe and both eastern and western North America; the T group consists of isolates mostly from hemlock from western North America. Results provide a benchmark for comparison of other isolates of these fungi and indicate the need for additional sampling and comparative research of these pathogens.