In Malus × domestica (Rosaceae) the product of each SFBB gene (the pollen component of the gametophytic self-incompatibility (GSI) system) of a S-haplotype (the combination of pistil and pollen genes that are linked) interacts with a sub-set of non-self S-RNases (the pistil component), but not with the self S-RNase. To understand how the Malus GSI system works, we identified 24 SFBB genes expressed in anthers, and determined their gene sequence in nine M. domestica cultivars. Expression of these SFBBs was not detected in the petal, sepal, filament, receptacle, style, stigma, ovary or young leaf. For all SFBBs (except SFBB15), identical sequences were obtained only in cultivars having the same S-RNase. Linkage with a particular S-RNase was further established using the progeny of three crosses. Such data is needed to understand how other genes not involved in GSI are affected by the S-locus region. To classify SFBBs specificity, the amino acids under positive selection obtained when performing intra-haplotypic analyses were used. Using this information and the previously identified S-RNase positively selected amino acid sites, inferences are made on the S-RNase amino acid properties (hydrophobicity, aromatic, aliphatic, polarity, and size), at these positions, that are critical features for GSI specificity determination.