Self-incompatibility is found in approximately 40% of flowering plant species and in at least 100 families. However, as the largest angiosperm family, there are only in 10% of orchid species predicted the presence of such mechanisms, of which might be gametophytic self-incompatibility, while the majority (72%) of Dendrobium, one of the largest Orchidaceae genus, show self-incompatibility. Studies of molecular characterization of the S-determinants of gametophytic self-incompatibility are well advanced in one group, which is Solanaceae, Rosaceae and Plantaginaceae S-RNase lineage (SLFs/S-RNase). In this study, we first identified two SI species using fluorescence microscopy. Then, we identify S-RNase and SSK1 like genes in the different transcriptomes tissues of Dendrobium longicornu and Dendrobium chrysanthum and genomes of Phalaenopsis equestris, Dendrobium. catenatum, Vanilla shenzhenica and Apostasia shenzhenica. Finally, the results of sequence characterization, phylogenetic analyses and specific expression in tissues revealed that none of the genes were the S-determinants. Therefore, there might be new S-determinants in Orchidaceae. So, to characterize the genes, 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. What’s more, RNase based GSI might have evolved after the split of the monocots and dicots and before the split of the Asteridae and Rosidae.