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The genus <i>Pyrus</i> is characterized by an <i>S</i>-RNase-based gametophytic self-incompatibility (GSI) system, a mechanism that promotes outbreeding and prevents self-fertilization. While the <i>S</i>-genotype of the most widely known pear cultivars was already described, little is known on the <i>S</i>-allele variability within local accessions. The study was conducted on 86 accessions encompassing most of the local Sicilian varieties selected for their traits of agronomic interest and complemented with some accessions of related wild species (<i>P. pyrifolia</i> Nakai, <i>P. amygdaliformis</i> Vill.) and some national and international cultivars used as references. The employment of consensus and specific primers enabled the detection of 24 <i>S</i>-alleles combined in 48 <i>S</i>-genotypes. Results shed light on the distribution of the <i>S</i>-alleles among accessions, with wild species and international cultivars characterized by a high diversity and local accessions showing a more heterogeneous distribution of the <i>S</i>-alleles, likely reflecting a more complex history of hybridization. The <i>S</i>-allele distribution was largely in agreement with the genetic structure of the studied collection. In particular, the “wild” genetic background was often characterized by the same <i>S</i>-alleles detected in <i>P. pyrifolia</i> and <i>P. amygdaliformis</i>. The analysis of the <i>S</i>-allele distribution provided novel insight into the contribution of the wild and international cultivars to the genetic background of the local Sicilian or national accessions. Furthermore, these results provide information that can be readily employed by breeders for the set-up of novel mating schemes.