Two distinct groups (A and B) of the conifer shoot blight and canker pathogen Sphaeropsis sapinea have been recognized on pine hosts in the northcentral United States. Their occurrence in a collection of isolates from much more diverse hosts and locations was studied by analysis of RAPD markers and isozymes. Seventy-nine isolates were used for RAPD marker analysis, and 37 for isozyme analysis, with 33 isolates in common. Hosts included 19 species in Pinus and species of Cedrus, Larix, Picea, Pseudotsuga, located in Africa, Australasia, Europe, and North America. Relationships among these isolates were determined using cluster analyses of presence or absence data for each amplification product or isozyme. Analyses of either RAPD marker or isozyme data placed most isolates in a group that contained previously characterized RAPD marker group A isolates. There was no strong indication of either host or geographic clustering within this highly similar, widely distributed group. Analysis of RAPD marker data placed several other isolates in a separate group that contained previously characterized RAPD marker group B isolates. This is the first report of isolates from hosts other than Pinus banksiana or P. resinosa, and from outside Michigan, Minnesota, or Wisconsin, that have been characterized as members of the RAPD marker group B.