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Genetic maps provide the foundation for QTL mapping of important traits of crops. As a valuable food and forage crop, rye (Secale cereale L., RR) is also one of the tertiary gene sources of wheat, especially wild rye, Secale cereale subsp. segetale, possessing remarkable stress tolerance, tillering capacity and numerous valuable traits. In this study, based on the technique of specific-locus amplified fragment sequencing (SLAF-seq), a high-density single nucleotide polymorphism (SNP) linkage map of the cross-pollinated (CP) hybrid population crossed by S. cereale L (female parent) and S. cereale subsp. segetale (male parent) was successfully constructed. Following preprocessing, the number of 1035.11 M reads were collected and 2425800 SNP were obtained, of which 409134 SNP were polymorphic. According to the screening process, 9811 SNP markers suitable for constructing linkage groups (LGs) were selected. Subsequently, all of the markers with MLOD values lower than 3 were filtered out. Finally, an integrated map was constructed with 4443 markers, including 1931 female mapping markers and 3006 male mapping markers. A major quantitative trait locus (QTL) linked with spike length (SL) was discovered at 73.882 cM on LG4, which explained 25.29% of phenotypic variation. Meanwhile two candidate genes for SL, ScWN4R01G329300 and ScWN4R01G329600, were detected. This research presents the first high-quality genetic map of rye, providing a substantial number of SNP marker loci that can be applied to marker-assisted breeding. Additionally, the finding could help to use SLAF marker mapping to identify certain QTL contributing to important agronomic traits. The QTL and the candidate genes identified through the high-density genetic map above may provide diverse potential gene resources for the genetic improvement of rye. [ABSTRACT FROM AUTHOR]