Lesion mimic mutants are ideal for studying disease resistance and programmed cell death photosynthesis in plants to improve crop yield. A novel light intensity-dependent lesion mimic mutant was obtained from the wheat variety Chuannong 16 (CN16) by ethyl methane sulfonate treatment. The mutant exhibited a lesion phenotype with degenerated chloroplast structure, decreased chlorophyll content, increased level of reactive oxygen species, and increased resistance to stripe rust and powdery mildew. A set of near-isogenic lines (NILs, NILlm5: without lesion spots; NILLm5: with lesion spots) was developed. Phenotypic analysis showed that significant differences were detected in plant height and flag leaf length, while not in spikelet number per spike, flag leaf width, effective tiller, spike length and spike extension length between the NILs. Genetic analysis indicated that the lesion phenotype was controlled by a novel single semi-dominant nuclear gene Lm5. It was further fine mapped in a 1.28 Mbp interval between markers KASP-5825 and KASP-9366 on chromosome arm 2AL containing 17 high-confidence candidate genes. Additionally, transcriptome and metabolome analysis of the NILs showed that differentially expressed genes and accumulated metabolites were mostly enriched in porphyrin and chlorophyll metabolism, glyoxylate and dicarboxylate metabolism and propanoate metabolism. Morphological observation and wheat germ agglutinin analysis of the NILs suggested that the leaves of NILlm5 had extensive growth of stripe rust mycelium within the leaves, while the infection of mycelia in the leaves of NILLm5 was almost invisible. Altogether, this study laid an important foundation for analyzing the formation mechanism of lesion spots and resistance mechanism of stripe rust.