Extensive survey of the S-locus diversity of plant species with RNase-based gametophytic self-incompatibility has failed to identify neutral variation segregating within S-allele specificities. Although this is the expected result according to population genetics theory, it conflicts with recent models of S-allele evolution, which suggest that new specificities might arise by a continuous process of subtle changes that individually do not alter the specificity of the S-genes, but whose cumulative effects result in new S-allele functions. Genomic analysis of S-RNase sequences associated with the S104 (=S4, =Sb) allele of European pear (Pyrus communis L.) cultivars yielded two distinct variants (named herein S104-1 and S104-2) that differed at five nucleotide positions within the open reading frame, two of which resulted in changes in the predicted protein sequence. Test-cross experiments indicated that the S-alleles associated with the S104-1 and S104-2RNases exhibit the same pollen and pistil functions, suggesting that they are two neutral variants segregating within the S104 haplotype of European pear. These allelic forms might represent transitional states in the process of generating new specificities in the species, in accordance with models that predict S-function transition through neutral intermediates. This possibility was further evaluated through the pattern of molecular evolution of functionally distinct European pear S-RNases, which indicated that most recent S-allele diversification in this species proceeded in the absence of adaptive selective pressure.