The enoki mushroom (Flammulina filiformis) is one of the most important and popular edible mushrooms commercially in China. However, traditional mushroom cultivar identification is challenging due to poor accuracy, heavy workloads, and low reproducibility. To overcome this challenge, we developed a method for identifying F. filiformis strains using multiple nucleotide polymorphism sequencing (MNP-seq). This involved screening 179 universal MNP markers based on whole-genome sequencing data, constructing an MNP sequence library, and performing multiplex PCR amplification and high-sequencing. We further screened 69 core MNP markers and used them to build a neighbor-joining (NJ) phylogenetic tree of 232 cultivated and wild strains. Our analysis showed that all cultivars could be accurately separated by computing genetic similarity values and that the cultivars could be separated into 22 distinct evolutionary pedigrees. The specific value of genetic similarity can be used as the standard to distinguish F. filiformis cultivars, however, it needs to be comprehensively defined by the additional phenotype and biological characteristics of those strains in the future work.