The rapid growth of aquaculture is driven by the increasing demand for fish which provide essential nutrients for balanced health. Empurau is a highly-sought freshwater fish with high market value due to its unique flesh taste, making it one of the most famous species among the aquaculture industry. However, the main challenge of the industry remains as the infectious disease outbreak with Pseudomonas species as one of the most threatening fish pathogens while residing a wide range of environments. Given antibiotics administration as the common solution to disease outbreaks, contributing to the worsening of antimicrobial resistance, the development of disease-resistant broodstock through the identification of SNP markers appeared as one of a promising strategy. In this study, a total of 1,048,576 SNP markers were identified via whole genome pooled sequencing on samples treated with LD 50, forming resistant and susceptible group. Multiple tests (pairwise F ST test, CMH test and FE test) and visualization had been conducted to screen and select candidate SNP markers for further validation using T-plex ARMS real-time PCR assay. The genotyping results on the selected candidate markers were confirmed using Sanger sequencing. Statistical analysis had been performed to validate the significance of the candidate marker. The successful validation of 19,564 G/C SNP markers is expected to advance research on disease-resistant association SNPs and support their integration into breeding programs, enabling the use of genomic information to optimize artificial selection in empurau breeding.