Lettuce has been commonly associated with the contamination of human pathogens, such as Escherichia coli O157:H7 (hereafter O157:H7), which has resulted in serious foodborne illnesses. Contamination events may happen throughout the farm-to-fork chain, when O157:H7 colonizes edible tissues and closely interacts with the plant. Environmental conditions have a significant impact on many plant-microbe interactions; however, it is currently unknown whether temperature affects O157:H7 colonization of the lettuce phyllosphere. In this study, we investigated the relationship between elevated growth temperatures, O157:H7 persistence, and lettuce head growth using 25 lettuce genotypes. Plants were grown under optimal or elevated temperatures for 3.5 weeks before being inoculated with O157:H7. The bacterial population size in the phyllosphere and lettuce head area was estimated at 0- and 10-days postinoculation (DPI) to assess bacterial persistence and head growth during contamination. We found that growing temperature can have a positive, negative, or no effect on O157:H7 persistence depending on the lettuce genotype. Furthermore, temperature had a greater effect on head area size than the presence of O157:H7. The results suggested that the combination of plant genotype and temperature level is an important factor for O157:H7 colonization of lettuce and the possibility to combine desirable food safety traits with heat tolerance into the lettuce germplasm.