Genetic diversity and relationships among 21 accessions of Secale L., including three species and 10 subspecies, were evaluated using RAMP markers. Forty-one out of 80 (50.5%) RAMP primers, which produced clear and polymorphic bands, were selected for PCR amplification of genomic DNA. A total of 446 bands were amplified from the 41 primers, and 428 of these bands (about 96%) were polymorphic. Three to 19 polymorphic bands could be amplified from each primer, with an average of 10.4 bands. The RAMP-based genetic similarity (GS) values among the 21 Secale accessions ranged from 0.266 to 0.658, with a mean of 0.449. A high level of genetic variation was found between or within the wild populations and the cultivars. Based on the GS matrix, a dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA). All 21 accessions could be distinguished by RAMP markers. Clustering results showed that the genetic diversity of Secale based on RAMP markers was correlated with geographical distribution. Six rye cultivars, originating from Poland, Portugal, Mexico, Hungary, Armenia and Ukraine, were clustered into one group. The six countries are all located in the transitional region of broad-leaf forests between maritime and continental temperate zones, with narrow latitude span. In comparison, the other five cultivars from countries scattered over a region with large latitude span were distributed within different groups or subgroups. Genetic relationships based on RAMP markers had great deviation from the original taxonomy. Some subspecies of the same species were distributed within different groups, while some accessions of different species were closely clustered into one subgroup. These results suggest that RAMP markers could be an effective technique for detecting genetic diversity among Secale and give some useful information about its phylogenic relationships.