•cDNA and genomic DNA sequences of the novel S59 allele were determined.•S59-specific and S10-specific PCR detection methods were developed for rapid identification from genomic DNA.•Self-incompatibility genotypes of over twenty accessions were characterized. Apples (Malus × domestica Borkh.) are an economically important crop in many temperate growing regions around the world. Apple being characterized by gametophytic self-incompatibility (GSI) requires cross-pollination with compatible apple pollen during bloom to achieve sufficient fruit set for commercial production. For this reason, in commercial orchards it is common practice to plant pollinizer trees, which can be either crabapples or different apple cultivars, at a density of 5–10%. Crabapple trees, in addition to their ornamental traits, are valued as a source of genetic diversity for pollinating apple orchards. The genetics underlying cross-compatible responses among crabapples and domesticated apples, especially recently released cultivars, have been largely understudied. In this study, we characterized one novel S-RNase allele from the crabapple Malus ‘Doubloons’, named S59, and report an allele-specific PCR method for detection of this allele from genomic DNA. Further, we characterized the self-incompatibility genotypes (S-genotypes) of over twenty previously unreported Malus accessions, including some recent releases from the breeding programs of the University of Minnesota and Washington State University. The results of this work aim to provide new information about cross-compatibility of cultivars and pollinizers and may be used to aid parent selection in apple breeding programs, as well as pollinizer selection for the commercial orchard.