Ophiostoma novo-ulmi, the principal agent of Dutch elm disease, has recently replaced another species of Dutch elm disease pathogen, O. ulmi, across much of the Northern Hemisphere. Field inoculations of the moderately resistant elms Ulmus procera and Ulmus × Commelin were carried out with progeny of a genetic cross between AST27, a Eurasian (EAN) O. novo-ulmi isolate with an unusually low level of pathogenicity, and H327, a highly aggressive EAN isolate. These confirmed the results of a previous study that indicated that the difference in phenotype was controlled by a single nuclear gene. This pathogenicity gene, designated here Pat1, is the first putative pathogenicity gene to be identified in O. novo-ulmi. In a bulked segregant analysis, involving 80 random primers, 10 RAPD (random amplified polymorphic DNA) markers were identified linked to Pat1. Linkage distances between these markers and Pat1 were confirmed by genetic analysis of all individual progeny. Five RAPD amplicons identified in AST27 were O. ulmi and not O. novo-ulmi specific amplicons, and of these two were linked to Pat1. This suggests that the Pat1 allele conferring unusually low aggressiveness in AST27 may have been acquired from O. ulmi via introgression. When RAPD marker OPK31050, linked to Pat1, was used as a probe for Southern hybridization with electrophoretically separated chromosomes of O. novo-ulmi and O. ulmi, the results indicated that Pat1 is located on a 3.5 Mb chromosome previously designated chDNA II.