The fungus Blumeria graminis f. sp. tritici (Bgt) caused powdery mildew is one of the destructive diseases of wheat. Resistance genes are the fundamental of disease resistance breeding to prevent wheat from powdery mildew caused damages. Aegilops longissima (2n=2x=14, SlSl), a wild relative of wheat, is an important gene pool of resistance to wheat diseases and pests. We previously identified a novel powdery mildew resistance gene Pm6Sl on Aegilops longissima chromosome 6Sl#3, and mapped the gene to a distal interval of 42.8 Mb in the long arm of 6Sl#3. Here, we reported the fine mapping and candidate gene exploration of gene Pm6Sl. A total of 34 6Sl#3-specific markers in the interval were designed based on the reference sequences of Ae. longissima, and 105 ph1b-induced homoeologous recombinants of 6Sl#3 were identified with recombined points in Pm6Sl-harboring interval. By integration of marker analyses and resistance assays using Bgt isolate E26 of those 6Sl#3 recombinants, the gene Pm6Sl was further mapped to a 0.31 Mb interval with eight key recombinants. Based on the reference genome of Ae. longissima TL05, thirteen predicted genes were located in the interval, of which, only four genes were annotated as disease resistance proteins, and the remaining nine genes encoded proteins of unknown functions or no annotations. Sequencing of these four genes in ten EMS-derived loss-of-resistance mutants revealed eight mutations only in a CNL (CC-NBS-LRR) gene but not in other three genes. Among these eight mutants, four had single-nucleotide variants that resulted in missense mutations and the remaining four led to premature stop codons. Therefore this CNL gene were considered as the putative candidate gene of Pm6Sl for further BSMV-VIGS and transgene verification. Besides, we identified several wheat-Ae. longissima recombinants with tiny 6Sl segments residing Pm6Sl, and diagnostic markers for marker-assistant selection of the gene. This study lays a foundation for the cloning and deployment of Pm6Sl, and understanding of resistance mechanism of the gene.