Three long terminal repeat (LTR) sequences of Ty1-copia retrotransposons were identified in cucumber (Cucumis sativus L.) and named Tcs 1, Tcs 2, and Tcs 3. A sequence-specific amplification polymorphism (SSAP) marker system based on these LTR sequences displayed a higher level of polymorphism than AFLPs in cucumber. This marker system could also detect loci in other Cucumis species for genetic diversity analysis. The three Tcs LTRs existed within the exons of genes because of the effective amplification band patterns from the cDNA templates. The potential usefulness of the SSAP marker system in studies of the evolution of genes or genomes was verified after exploring loci changes in first and second generations of a synthetic allotetraploid in Cucumis. This study is the first report of the development of a retrotransposon-based marker system and the SSAP technique in cucurbits.