The most popular retrotransposon-based molecular marker system at the present time is the sequence specific amplification polymorphism (S-SAP) system, which is widely used for various applications including phylogenetic analysis. In this study, we successfully developed a new S-SAP marker system based on Ty1-copia retrotransposon in apple genome. Among 34 primer combinations used to develop the S-SAP marker system, seven primer combinations produced high-quality and reliable banding patterns. A total of 84 polymorphic bands from 588 amplified bands were detected in 27 Delicious sport cultivars, with a polymorphic ratio of 8.1% and genetic coefficient ranged from 0.73 to 0.90. Phylogenetic analysis showed that genetic coefficient ranged from 0.42 to 0.94 for 15 apple sport cultivars, and the variety line was distinguished based on genetic coefficient 0.80. The available evidences proved S-SAP marker had high level of polymorphism in apple accessions, efficiently applied to genetic diversity, systematic evolution and identification of variety.