Rosy apple aphid (Dysaphis plantaginea), is one of the major insect pests of apple, causing serious physical and economic damage to fruit production. A dominant resistance gene Dp-fl was previously mapped at the bottom of linkage group LG8 from the cultivar ‘Florina’, linked to the SSR CH01h10. The development of additional genetic markers mapping closer to Dp-fl was needed to position the gene accurately and to improve the effectiveness of marker-assisted breeding (MAB). The aims of this study were to identify single nucleotide polymorphisms (SNPs) in the region of Dp-fl and to position these SNPs relative to Dp-fl. To generate a fine map of the Dp-fl interval, a total of 191 plants segregating for resistance and derived from four different populations were tested with temperature-switch PCR (TSP) markers developed for SNPs located in the region of CH01h10. All the plants were phenotypically evaluated for aphid resistance and those data compared with the genetic data. These efforts resulted in positioning the Dp-fl resistance locus in a genetic interval corresponding to a physical distance of about 330 kb on the ‘Golden Delicious’ genome. The new markers were tested on several apple founder cultivars in order to test the specificity of the SNPs and, thus, the best markers for the MAB were identified. Finally, the 330-kb interval was analyzed for the identification of coding sequences and putative candidate genes for D. plantaginea resistance were identified.