Black cherry (Prunus serotina) is a tetraploid species with a commercial interest in Ecuador, however, it has a mechanism that decreases productivity of fruit trees. One of the genes that controls this mechanism (gametophytic self-incompatibility) is the S-RNase, therefore, its alleles define which cultivars are compatible. S-RNase gene has alleles that have the same size but different sequence, thus, nanopore sequencing was a suitable approach to discriminate these alleles. In this investigation, the MinION device was used to distinguish the S-RNase alleles from 12 samples of black cherry, by using a multiplex sequencing approach and a self-design bioinformatic analysis pipeline.