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A total of 1027 genotypes of wild Lactuca spp. (L. serriola, L. saligna, and L. aculeata), originating from Israel, Jordan, East Turkey, Armenia, Kazakhstan, China, and various other countries in Europe were screened for resistance to the lettuce downy mildew (Bremia lactucae Regel). The genotypes of L. serriola (N = 962); L. saligna (N = 43); and L. aculeata (N = 22) were tested at the seedling stage with two isolates of B. lactucae (race Bl-21 and Isr-74) isolated from cultivated lettuce in Italy and Israel, respectively. Altogether, the isolates possess virulence against 17 out of 20 Dm genes/R-factors composing the established differential set of lettuce lines/varieties. They could therefore be used for preliminary detection of the presence of new resistance factors. A total of 83 and three genotypes of L. serriola and L. saligna, respectively, were resistant to infection by each of the two isolates alone as well as to the infection by a mix of the two isolates. These genotypes probably carry previously unknown resistance, and therefore could be suitable for exploitation by breeders and further detailed research. The wild progenitor of lettuce, L. serriola, and related species harbor significant untapped genetic resources for lettuce improvement.