The invention discloses an MNP core primer combination for molecular identification of DNA varieties of nelumbo nucifera L. and an application of the MNP core primer combination. On the basis of re-sequencing data of the nelumbo nucifera L., 525 pairs of MNP core primer combinations for detecting polymorphic MNP marker sites of the nelumbo nucifera L. are screened by combining a high-throughput sequencing technology, and the nucleotide sequences of the combinations are as shown in SEQ ID No.1-SEQ ID No.1050. The 525 pairs of MNP site detection primers provided by the invention can be compatible with a next-generation sequencing platform, have the characteristics of high efficiency, accuracy, high reproducibility and the like, and meet the requirements of standard fingerprint data construction of the nelumbo nucifera L. The primer combination can be used for identifying a to-be-detected nelumbo nucifera L.sample and a control variety, can accurately identify the authenticity of the to-be-detected nelumbo nucifera L. sample, can be applied to the aspects of constructing a nelumbo nucifera L. DNA fingerprint database, detecting the authenticity of the nelumbo nucifera L. variety, analyzing genetic diversity of nelumbo nucifera L. germplasm resources or breeding nelumbo nucifera L.molecules and the like, and provides technical support for breeding nelumbo nucifera L. molecules and analyzing seed resources. The breeding period of the new nelumbo nucifera L.variety is accelerated.