In this study, S-RNase-homologous genes expressed in styles were isolated from a gametophytic self-incompatible citrus cultivar, ‘Hyuganatsu’. Sweet orange and clementine genome databases were searched to identify 13 ribonuclease T2 (T2 RNase) genes. Further blast searches using citrus EST databases were conducted with these 13 sequences as queries to obtain five additional EST sequences. Known T2 RNase genes, including the S-RNases of Rosaceae, Solanaceae, and Plantaginaceae were retrieved from the public database. All data collected from the databases were combined to make a dataset for phylogenetic analysis. From the phylogenetic tree, 10 citrus sequences were found to be monophyletic to the clade of S-RNases. Degenerate primers were designed from these genes to identify similar sequences in cDNA derived from ‘Hyuganatsu’ styles. RT-PCR and 3' and 5' RACE resulted in the isolation of three S-RNase-like sequences; these sequences were further characterized. Pistil-specific gene expression was confirmed for all sequences by RT-PCR; Citrus tamurana RNase1 (CtRNS1) and RNase3 (CtRNS3) had basic pI values (> 8) and molecular weights of approximately 25 kDa, consistent with S-RNase features of other families. The distribution of CtRNS1, CtRNS2, and CtRNS3 was investigated using 10 citrus cultivars. CtRNS1 and CtRNS2 did not correspond to any S-haplotypes in the 10 cultivars, while CtRNS3 matched the citrus’ S₁-haplotype.