After-cooking darkening (ACD), a gray-black discoloration, is one of the key quality defects of the potato. Previous work using a diploid population 13610 identified 14 single nucleotide polymorphism (SNP) markers, located on eight chromosomes, which have the strongest association with ACD. Seven of these markers are located in four major quantitative trait loci (QTL) on chromosomes 2, 4, 6, and 10. It is hypothesized that these 14 markers would also have significant relationship to ACD in other breeding populations. Therefore, their effects in another diploid population 14946 were analyzed by amplicon genotyping using high-resolution DNA melting (HRM). Eleven of the 14 SNP markers were confirmed to be significantly associated with ACD. Six of the 11 markers were located in the four major QTL regions. In addition, unlabelled probe assays using HRM were performed on a group of clones in the tetraploid population 14945. Five from the 14 markers were analyzed, and three showed significant relationship to ACD. The methodology established and the markers identified in this study could benefit breeding programs for developing lower ACD varieties.