Little is known regarding the genes, compounds and physiological alternations that take place upon infection of black knot disease. This research aimed to unravel the genetic mechanism responsible for the resistance of Japanese plum (Prunus salicina L.) trees against black knot (Apiosporina morbosa Schwein.) using a Genome-Wide Association Study. Genotyping by Sequencing (GBS) was combined with a phenotyping system to analyze 200 genotypes of mixed origin. Population stratification identified four subpopulations, and the Fixed and Random Model Circulating Probability Unification (FarmCPU) algorithm was used for this analysis. Nineteen single nucleotide polymorphisms (SNPs) significantly associated with black knot disease resistance were discovered across five chromosomes. Linkage disequilibrium analysis identified 55 genes near these SNPs, with eight genes related to plant defense, immunity, and biotic stress response. One SNP mutation was found in the 5' untranslated region of a gene regulating the first enzyme in phenylpropanoid biosynthesis. The results provide valuable insights into the genetic mechanisms behind BLACK KNOT disease resistance in Japanese plum and identifies potential markers for use in molecular breeding.