Isolates of Cylindrocladium floridanum obtained from diseased roots of white and black spruce seedlings and surrounding soil in bareroot nurseries in Ontario, Minnesota and Wisconsin area were compared by mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) restriction fragment length polymorphisms (RFLPs), human minisatellite DNA fingerprints and polymerase chain reaction. Nucleotide sequences of rDNA ITS regions of selected isolates were also performed. Based on the nuclear DNA fingerprinting patterns, these isolates could be divided into three groups, namely S1, S2 and S3. Isolates of S1 and S2 showed the same mtDNA RFLPs which were slightly different from that of S3. Isolates representing each of the three groups showed an identical nucleotide sequence of rDNA ITS regions. These data suggest that all the isolates tested belong to the same species which may be divided into three biotypes or populations.