Vanilla planifolia, the prominent cultivated vanilla species, exhibits a distinct form of endoreplication specific to orchids, termed partial endoreplication. This process results in asymmetrical nuclei, composed of an endoreplicated fraction and a non endoreplicated fraction of the genome. Despite a genome assembly of Vanilla planifolia encompassing 3.4 Gb of sequences, approximately two-thirds of these sequences remain unanchored within the species chromosomes. These unanchored sequences mainly consist of sequences from the non-endoreplicated fraction of the genome, posing challenges to assembly due to its low sequencing depth compared to the endoreplicated one. . This study pioneers genotyping efforts concentrating on the development of markers, specifically microsatellites, tailored to the non-endoreplicated fraction of the genome. Five microsatellites were identified as polymorphic within the CR0040 self-fertilised population of 119 lines. These markers were subsequently integrated into an existing genetic map, with four out of five markers positioned relatively distant from GBS-SNP markers in their respective linkage groups. Notably, one marker was successfully allocated to its linkage group, corresponding to chromosome A10. These microsatellites, designed in the non-endoreplicated fraction of the genome, hold potential for future applications in cytogenetic, diversity, and phylogenetic studies to study the mechanism of partial endoreplication.