Nucellar seedlings of polyembryonic varieties are utilized as rootstocks hence distinguishing nucellar from zygotic seedlings in the polyembryonic kernel is crucial for obtaining clonal rootstock material. The current study aimed to identify the origin of multiple seedlings in the polyembryonic Mango cultivars Vellaikulamban and Olour with monoembryonic reference Totapuri. Morphological studies on embryos’ position, fresh weight, length, and width of kernel embryos were recorded. Twelve markers were used to identify the origin of seedlings. The third position embryo in the polyembryonic kernels exhibited maximum fresh weight, length, and width followed by 4th and 2nd position embryos. However, the average embryos per kernel were maximum in Olour. Analysed PCR products were subjected to gene scan analysis and data was compared through the Neighbour Joining method. Eleven markers showed polymorphism in the differentiation of nucellar and zygotic seedlings. Based on the genetic dissimilarity, the genetically variant zygotic seedlings of polyembryonic genotypes (VK-3C, OL-3C, OL-4D, Tota-1) were grouped with monoembryonic maternal Totapuri (Tota-M) in the same cluster while those nucellar originated seedlings of Vellaikulamban and Olour were grouped with their respective maternal parent (VK-M, OL-M). The current study provides a basic understanding of prior seedling identification for the selection of clonal rootstock for propagation.