Abstract Background Fabaceae species are important in agronomy and livestock nourishment. They have a long breeding history, and most cultivars have lost self-incompatibility (SI), a genetic barrier to self-fertilization. Nevertheless, to improve legume crop breeding, crosses with wild SI relatives of the cultivated varieties are often performed. Therefore, it is fundamental to characterize Fabaceae SI system(s). We address the hypothesis of Fabaceae gametophytic (G)SI being RNase based, by recruiting the same S-RNase lineage gene of Rosaceae, Solanaceae or Plantaginaceae SI species. Results We first identify SSK1 like genes (described only in species having RNase based GSI), in the Trifolium pratense , Medicago truncatula, Cicer arietinum, Glycine max , and Lupinus angustifolius genomes. Then, we characterize the S -lineage T2-RNase genes in these genomes. In T. pratense , M. truncatula, and C. arietinum we identify S-RNase lineage genes that in phylogenetic analyses cluster with Pyrinae S-RNases . In M. truncatula and C. arietinum genomes, where large scaffolds are available, these sequences are surrounded by F-box genes that in phylogenetic analyses also cluster with S- pollen genes. In T. pratense the S-RNase lineage genes show, however, expression in tissues not involved in GSI. Moreover, levels of diversity are lower than those observed for other S - RNase genes. The M. truncatula and C. arietinum S-RNase and S- pollen like genes phylogenetically related to Pyrinae S -genes, are also expressed in tissues other than those involved in GSI. To address if other T2-RNases could be determining Fabaceae GSI, here we obtained a style with stigma transcriptome of Cytisus striatus , a species that shows significant difference on the percentage of pollen growth in self and cross-pollinations. Expression and polymorphism analyses of the C. striatus S-RNase like genes revealed that none of these genes, is the S- pistil gene. Conclusion We find no evidence for Fabaceae GSI being determined by Rosaceae, Solanaceae, and Plantaginaceae S-RNase lineage genes. There is no evidence that T2-RNase lineage genes could be determining GSI in C. striatus . Therefore, to characterize the Fabaceae S -pistil gene(s), expression analyses, levels of diversity, and segregation analyses in controlled crosses are needed for those genes showing high expression levels in the tissues where GSI occurs.