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Fusarium species can cause severe root disease and damping-off in conifer nurseries. Fusarium inoculum is commonly found in most container and bareroot nurseries on healthy and diseased seedlings, in nursery soils, and on conifer seeds. Isolates of Fusarium spp. can differ in virulence; however, virulence and colony morphology are not correlated. Forty-one isolates of Fusarium spp., morphologically indistinguishable from F. oxysporum, were collected from nursery samples (soils, healthy seedlings, and diseased seedlings). These isolates were characterized by amplified fragment length polymorphism (AFLP) and DNA sequencing of nuclear rDNA (internal transcribed spacer including 5.8s rDNA), mitochondrial rDNA (small subunit [mtSSU]), and nuclear translation elongation factor I-alpha. Each isolate had a unique AFLP phenotype. Out of 121 loci, 11 1 (92%) were polymorphic; 30 alleles were unique to only highly virulent isolates and 33 alleles were unique to only isolates nonpathogenic on conifers. Maximum parsimony and Bayesian analyses of DNA sequences from all three regions and the combined data set showed that all highly virulent isolates clearly separated into a common clade that contained F. commune, which was recently distinguished from its sister taxon, F. oxysporum. Interestingly, all but one of the nonpathogenic isolates grouped into a common clade and were genetically similar to F. oxysporum. The AFLP cladograms had similar topologies when compared with the DNA-based phylograms. Although all tested isolates were morphologically indistinguishable from F. oxysporum based on currently available monographs, some morphological traits can be plastic and unreliable for identification of Fusarium spp. We consider the highly virulent isolates to be F. commune based on strong genetic evidence. To our knowledge, this is the first reported evidence that shows F. commune is a cause of Fusarium disease (root rot and damping-off) on Douglas-fir seedlings. Furthermore, several AFLP genetic markers and mtSSU sequences offer potential for development of molecular markers that could be used to detect and distinguish isolates of F. oxysporum nonpathogenic to conifers and highly virulent isolates of F. commune in forest nurseries.