In plants, lesion mimic mutants (LMMs) develop apoptosis-like lesions or necrosis on the leaves in the absence of pathogen. Previous study showed that LMMs were related to enhanced resistance to a broad spectrum of diverse pathogen races. In this study, we found a small number of plants with a lesion mimic phenotype appeared in a segregating population obtained by crossing two normal green wheat strains, Xinong 509 and N07216. Then we analyzed the different hybridized combinations’ spot phenotypes of the consecutive progeny populations over seven cropping seasons. By combining genetic data, we inferred that the spotted leaf lesion mimic phenotype was controlled by a dominant gene designated TaSpl1, which was inhibited by two other dominant genes, designated TaSpl1-I1 and TaSpl1-I2. Using BSR-Seq and newly developed KASP-PCR markers, the TaSpl1 and TaSpl1-I1 loci in N07216 were mapped to the end of chromosomes 3DS and 3BS, respectively. The TaSpl1 was flanked by the markers KASP188 and KASP229. The spotted plants showed lower levels of stripe rust and powdery mildew than the normal plants. Compared with normal leaves, the differentially expressed genes in spotted leaves were significantly enriched in plant-pathogen interaction and endocytosis pathways. Our results are trying to redefine the genetic and regulatory models of reported spotted leaf genes, and maybe provide a novel disease-resistance breeding strategy by using the lesion mimic allele.