Walnut anthracnose induced by Colletotrichum gloeosporioides is a devastating disease that seriously threatens walnut cultivation. Screening novel resistance genes and exploring the molecular mechanisms are essential for disease-resistant genetic improvement of walnut. We conducted a genome-wide association studies of disease resistance traits based on the relative resistance index and single nucleotide polymorphisms (SNPs) obtained from 182 resequenced walnut accessions and 10 loci and corresponding candidate genes associated with resistance against C. gloeosporioides were identified. Then, through combined transcriptome analysis during C. gloeosporioides infection and qRT-PCR, we identified JrWDRC2A9 in SNP Chr13_36265784 loci and JrGPIAP in SNP Chr07_10106470 loci as two walnut anthracnose resistance genes. The validation of the disease resistance function of transgenic strains indicated that both JrWDRC2A9 and JrGPIAP promote walnut resistance to anthracnose. SNP Chr13_36265784 (A>G) is located in the coding region of JrWDRC2A9 causing a glutamine (JrWDRC2A9HapI) to arginine (JrWDRC2A9HapII). Allelic variation in the WD domain attenuates JrWDRC2A9-mediated resistance against C. gloeosporioides and the binding affinity of JrWDRC2A9 for JrTLP1. On the contrary, the allelic variation caused by SNP Chr07_10106470 (T>G) increased the walnut accessions resistance to C. gloeosporioides by promoting the expression level of JrGPIAP. Functional genomics revealed that JrGPIAP binds to the promoter of JrPR1L and activates its transcription, which is strengthened by the interaction between JrGPIAP and JrEMP24. These findings reveal the allelic variation in JrWDRC2A9 and JrGPIAP conferring resistance against C. gloeosporioides, providing a genetic basis for walnut disease resistance breeding in the future.