Forty-seven isolates of Verticillium albo-atrum, 35 from hop (Humulus lupulus), seven from lucerne (alfalfa, Medicago sativa) and five from four other hosts, were analysed for DNA polymorphisms. Restriction fragment length polymorphisms (RFLPs) were detected in ribosomal RNA genes (rDNA) using Southern hybridization. Polymorphisms in mitochondrial DNA (mtDNA) were detected in ethidium bromide stained gels after digestion of total genomic DNA with restriction enzymes which recognize four bases containing only G and C. Amplified polymorphic DNA (APD) was analysed using primers based on rDNA sequences from the intergenic spacer (IGS) and 25S regions. These data were used to construct phenograms using either squared Euclidean dissimilarity coefficients (SEDC) and cluster analysis, or unweighted pair grouping with arithmetic averaging (UPGMA). rDNA RFLPs revealed one group with 44 isolates, a second group with two atypical hop isolates, and a third group containing a single avirulent lucerne isolate. mtDNA RFLPs separated rDNA group one into two subgroups, one group containing 38 isolates from different hosts and the other containing all six virulent lucerne isolates. APD analysis divided the isolates into 16 phenotypes, 12 of which contained most of the hop isolates, but there was no correlation with origin, hop cultivar, pathogenicity or year of isolation. One APD phenotype contained all six virulent lucerne isolates, indicating the genetic differentiation between hop and lucerne isolates. Two further APD phenotypes coincided with the second atypical group containing two hop isolates and a distinct avirulent lucerne isolate, respectively. The three methods revealed that three isolates differed markedly from those of the main group.