Abstract: The present disclosure provides an identification method of two parents of a Nymphaea hybrid based on sequences of internal transcribed spacer (ITS) and matK, and belongs to the technical field of plant molecular identification. In the present disclosure, the identification method includes: based on Sanger sequencing, obtaining an ITS sequence of a nuclear genome fragment inherited from the two parents and a matK sequence of a chloroplast genome fragment inherited from a maternal line in the Nymphaea hybrid; subjecting ITS and matK sequences downloaded from a GenBank to strict screening, and establishing a database of the ITS and matK sequence of the Nymphaea; aligning ITS and matK sequences of the Nymphaea of the parents to be determined with respective database, and constructing a neighbor-joining tree based on a genetic distance; and checking specific loci to obtain information on male and female parents of the Nymphaea to be determined.