Leaf rust is among the most destructive diseases of wheat globally, incurring significant yield losses and serious economic damage. Characterization and fine mapping of genetic loci for leaf rust resistance can be beneficial for marker-assisted breeding. In the present study, we identified three stable quantitative trait loci (QTL) for adult-plant leaf rust resistance, designated QLr.caas-2AS, QLr.caas-2DS and QLr.caas-5AL, respectively, in a recombinant inbred line population derived from a Zhongmai 175 × Lunxuan 987 cross across four environments. Fine mapping of QLr.caas-5AL was subsequently conducted using a secondary population derived from 18 heterozygous recombinants and 19 Kompetitive allele-specific PCR (KASP) markers, which allowed the delimitation of the QTL to a 794.8-kb physical interval from 523.6 to 524.4 Mb based on the Chinese Spring reference genome v1.1. Eighteen high-confidence annotated genes were present in this physical interval, and three genes showing sequence polymorphisms and differential expression between parents were predicted as candidates for QLr.caas-5AL, based on genome and transcriptome sequencing analyses. A KASP marker for QLr.caas-5AL was successfully developed and validated to be significantly associated with leaf rust severity in a natural wheat population of 221 cultivars. The frequency of the resistance allele at this KASP marker locus was 48.0% in the wheat cultivar panel. These findings not only lay a robust foundation for map-based cloning of QLr.caas-5AL, but also provide an efficient molecular tool for marker-assisted selection in wheat breeding.