Lettuce bacterial leaf spot caused by Xanthomonas campestris pv. vitians is an aggressive disease that is difficult to control. So far there are no reports of the reaction of biofortified lettuce genotypes to different isolates of the bacteria. Thus, the objective was to evaluate the aggressiveness of X. campestris pv. vitians, as well as the reaction of biofortified lettuce genotypes to bacterial spot. Two experiments were performed in two distinct seasons (winter and summer), in greenhouse at the Vegetable Experimental Station of the Federal University of Uberlândia (UFU). The experimental design in both experiments was a randomized block design, in a factor scheme of 5 × 4 (five genotypes and four strains), with four repetitions. Were evaluated the severity and the area under the disease progress curve. In general, the biofortified lettuce 'Uberlândia 10000' was more resistant to most bacterial strains in the summer cultivation, and in the winter period UFU 'Crespa 206'. The commercial cultivar Robusta was the most susceptible to the strains during both seasons. The UFU E125 strain was the most aggressive for most genotypes in both seasons.