Eyespot, caused by Oculimacula yallundae and O. acuformis, is an economically important disease of wheat. Currently, two eyespot resistance genes, Pch1 and Pch2, are used in wheat breeding programs but neither provides complete control or prevents yield loss. Aegilops longissima is a distant relative of wheat and proven donor of genes useful for wheat improvement, including disease resistance. Forty A. longissima accessions and 83 A. longissima chromosome addition or substitution lines were evaluated for resistance to eyespot. Among the 40 accessions tested, 43% were resistant to O. yallundae, 48% were resistant to O. acuformis, and 33% were resistant to both. Addition or substitution lines containing chromosomes 1S1, 2S1, 5S1, and 7S1, and a 4S17S1 translocation were resistant to O. yallundae. Chromosomes 1S1, 2S1, 4S1, and 5S1 contributed to resistance to O. acuformis more than others. Chromosomes 1S1, 2S1, 5S1, and 7S1 provided resistance to both pathogens. This is the first report of eyespot resistance in A. longissima. These results provide evidence that genetic control of eyespot resistance is present on multiple chromosomes of the S1 genome. This research demonstrates that A. longissima is a potential new source of eyespot resistance genes that could broaden the genetic diversity for wheat improvement.