The S-RNase based gametophytic self-incompatibility (GSI) system is a genetic mechanism present in Solanaceae, Plantaginaceae, Rosaceae, and Rubiaceae, that prevents self-fertilization. This system emerged before the split between Asteridae and Rosidae, about 120 million years ago (MYa), and thus, it is expected to be present in many self-incompatible (SI) species belonging to these subclasses. Although the system is ancestral, within the Rosaceae family, there is evidence for convergent evolution. Namely, Malus and Prunus S-RNase and S-pollen genes belong to distinct gene lineages. Furthermore, in Fragaria lineage (that is ancestral to Malus and Prunus) there is evidence for the presence of the Prunus S-RNase and S-haplotype specific F-box (SFB) lineage only. To understand the ancestral Rosaceae S-locus region, putative T2-RNases were characterized from Rosa (Roseae) and Rubus (Rubeae) genomes. Phylogenetic analyses were performed to identify putative S-locus genes. Such data is fundamental to perform segregation experiments, that are crucial to find the functional S-RNase gene. To explore the hypothesis that different S-RNase lineage genes can be recruited for GSI, as observed in Rosaceae, we also characterized Fabaceae S-RNase lineage genes, in order to identify the functional S-RNase gene. Fabaceae is a Rosidae, and SI species, such as Trifolium pratense, are expect to present S-RNase based GSI. Phylogenetic analyses of S-RNase lineage genes in five Fabaceae genomes, including one from T. pratense (Tatra cultivar), revealed 14 of these genes. Those genes that cluster with Prunus and Malus S-RNases present levels of diversity and expression that are incompatible with their involvement in GSI specificity determination. In this work we annotated the S-RNase lineage genes from the genome of T. pratense Milvus B cultivar, that has been assembled into chromosomes. Levels of diversity and expression analyses were performed for T. pratense T2-RNase lineage genes, in order to address their involvement in GSI specificity determination.