Understanding pathogenic variation in plant pathogen populations is key for the development and use of host resistance for managing verticillium wilt diseases. A highly virulent defoliating (D) pathotype in Verticillium dahliae has previously been shown to occur only in one clonal lineage (lineage 1A). By contrast, no clear association has yet been shown for race 1 with clonal lineages. Race 1 carries the effector gene Ave1 and is avirulent on hosts that carry resistance gene Ve1 or its homologues. The hypothesis tested was that race 1 arose once in a single clonal lineage, which might be expected if V. dahliae acquired Ave1 by horizontal gene transfer from plants, as hypothesized previously. In a diverse sample of 195 V. dahliae isolates from nine clonal lineages, all race 1 isolates were present only in lineage 2A. Conversely, all lineage 2A isolates displayed the race 1 phenotype. Moreover, 900-bp nucleotide sequences from Ave1 were identical among 27 lineage 2A isolates and identical to sequences from other V. dahliae race 1 isolates in GenBank. The finding of race 1 in a single clonal lineage, with identical Ave1 sequences, is consistent with the hypothesis that race 1 arose once in V. dahliae. Molecular markers and virulence assays also confirmed the well-established finding that the D pathotype is found only in lineage 1A. Pathogenicity assays indicated that cotton and olive isolates of the D pathotype (lineage 1A) were highly virulent on cotton and olive, but had low virulence on tomato.