Abstract: Medicago truncatula is another whole genome sequenced species next to Arabidopsis thaliana and Oryza sativa. Studying genomics and genetics using cross-species markers developed from M. truncatula has become an important strategy for species with more complex genomes or for legumes with less well understood genome. M. lupulina and M. polymorpha are the most important annual medics in China. However, limited by molecular marker, little research has been done on genome scales in these species. Specific length amplification fragment sequencing (SLAF-seq) possesses significant advantages including development of markers with high throughput, high accuracy, low cost and time saving which has been successfully used in single nucleotide polymorphism (SNP) development in many species. In this study, 28.04×10⁶ reads were obtained from a collection of twelve of M. truncatula, M. lupulina and M. polymorpha accessions using SLAF-seq. The average sequencing depth was 17.44, and 276432 high-quality SLAFs were developed, among which 58748 SLAFs were polymorphic. Further 189133 cross-species SNPs were identified from these polymorphic SLAFs with minor allele frequencies (MAFs)>0.05. These SNPs can be used in genetic diversity, genetic linkage mapping and QTL mapping of important agronomic traits in annual medic. They will also convey benefits for the study of genome orders, system evolution, and comparative map construction.