Self-incompatibility (SI) is an important mechanism that prevents self-fertilization and inbreeding in flowering plants. The most widespread SI system utilizes S ribonucleases (S-RNases) and S-locus F-boxes (SLFs) as S determinants. In citrus, SI is ancestral, and Citrus maxima (pummelo) is self-incompatible, while Citrus reticulata (mandarin) and its hybrids are self-compatible (SC). Here, we identify nine highly polymorphic pistil-specific, developmentally expressed S-RNases from pummelo that segregate with S haplotypes in a gametophytic manner and cluster with authentic S-RNases. We provide evidence that these S-RNases function as the female S determinants in citrus. Moreover, we show that each S-RNase is linked to approximately nine SLFs. In an analysis of 117 citrus SLF and SFL-like (SLFL) genes, we reveal that they cluster into 12 types and that the S-RNases and intra-haplotypic SLF and SLFL genes co-evolved. Our data support the notion that citrus have a S locus comprising a S-RNase and several SLFs that fit the non-self-recognition model. We identify a predominant single nucleotide mutation, Sm-RNase, in SC citrus, which provides a ‘natural’ loss of function. We show that SI–SC transitions due to the Sm-RNase initially arose in mandarin, spreading to its hybrids and became fixed. Identification of an evolutionarily distant new genus utilizing the S-RNase-based SI system, >100 million years separated from the nearest S-RNase family, is a milestone for evolutionary comparative studies.