ABSTRACT Verticillium wilt, caused by Verticillium dahliae, poses a major threat to lettuce (Lactuca sativa) production in California. Incorporation of resistance into commercial lettuce cultivars offers the least expensive technique of sustaining production in infested areas. To test the breadth of the resistance identified in field experiments, a pair of susceptible ('Salinas' and 'Sniper') and resistant ('La Brillante' and 'Little Gem') lettuce cultivars were used as differentials and individually inoculated with 29 isolates of V. dahliae and two isolates of V. albo-atrum from several hosts, including lettuce, in replicated greenhouse experiments. The reactions of the four cultivars were determined based on the disease severity at maturity. None of the V. albo-atrum isolates or V. dahliae isolates from cruciferous hosts caused significant disease on lettuce. Both Salinas and Sniper were susceptible to many isolates of V. dahliae (21 of 23) from noncruciferous hosts, and the isolates varied in their overall virulence. However, of these, only three isolates caused significant disease on the resistant cvs. La Brillante and Little Gem. These three isolates also were distinct from the other V. dahliae isolates based on sequence data from the intergenic spacer (IGS) region of the nuclear ribosomal RNA gene, suggesting that they form a phylogenetically distinct subgroup that differs in virulence toward specific lettuce genotypes. Accordingly, isolates of V. dahliae virulent on all tested cultivars, including the resistant La Brillante and Little Gem, were designated as race 2, whereas those virulent only on the susceptible Salinas and Sniper were designated as race 1. Although a range of virulence among isolates has been described in other hosts, this is the first description of distinct virulence phenotypes in V. dahliae since a similar race structure was described in tomato in the 1960s.