Daphnia magna belongs to the Cladocera and plays an important role in the water ecosystem. With the intensification of water pollution, the wild population of D. magna has declined rapidly in recent years, and insufficient molecular markers have limited effective research and conservation of this species. In this research, 30 novel single nucleotide polymorphism (SNP) markers were developed in a cultivar of Daphnia magna and 12 wild Daphnia magna using restriction site-associated DNA sequencing (RAD-seq). The minor allele frequency, observed heterozygosity, and expected heterozygosity ranged from 0.115 to 0.721, 0.073 to 0.800, and 0.077 to 0.520, respectively. The PIC ranged from 0.071 to 0.403. Six loci showed significant deviations from the Hardy-Weinberg equilibrium after Bonferroni correction (p < 0.05).These newly developed polymorphic SNP markers for D. magna are of great significance in terms of the genetic breeding of D. magna, identification of wild and artificially domesticated species and conservation genetics research.