The self-incompatibility (S) genotypes of a collection of 65 Belgian accessions of wild cherry, selected within two populations and planted in a seed orchard, were determined using polymerase chain reaction (PCR) methods. Initially, DNA extracts were amplified with consensus primers that amplify across the second intron of the S-ribonuclease gene which shows considerable length polymorphism. The provisional genotypes deduced were checked with the appropriate allele-specific primers for the known alleles S1 to S16. Putative new alleles were subjected to PCR with consensus primers amplifying across the first intron. Six new alleles, S17 to S22, were thus indicated on the basis of the estimated lengths of the first and second intron PCR products. Examples of these alleles were partially sequenced and were indeed mutually distinct and different from the known alleles. The incompatibility genotypes of all 65 accessions were determined and one triploid individual was found. Seventeen alleles were detected in all. Allele frequencies differed between samples and the expected total number of alleles in the underlying populations was estimated. The wild cherry populations differed significantly with respect to allelic frequencies from sweet cherry cultivars; alleles S4 and S5, which are moderately frequent in sweet cherry, were absent from the wild cherry accessions. The knowledge of the S genotypes will be useful for studying the gene flow within the seed orchard and these approaches should also be informative in wild populations.