Daphnia magna belongs to the Cladocera order 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 our research, 26 novel microsatellite (SSR) markers were developed in an artificially domesticated of D. magna and 12 wild population of D. magna using restriction site-associated DNA sequencing (RAD-seq). The results showed that the observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.083 to 0.999 and 0.085 to 0.862, respectively. The PIC ranged from 0.368 to 0.805. These results indicate that the developed SSR marker is highly polymorphic. Nei’s genetic identity (H) ranged from 0.0926 to 0.3462, with a mean of 0.2233. Shannon’s Information index (I) ranged from 0.1333 to 0.4799, with an average of 0.3073; Shanxi province had the highest value and Hunan province had the lowest. Genetic distance and Nei’s genetic identity analysis, NJ tree diagram analysis, and PCoA analysis were conducted on populations of Daphnia magna from different regions. An analysis of population genetic structure divided the test D. magna samples into two major groups. There was no absolute correlation between the gene distribution of D. magna and its geographic origin. These results indicate that the genetic diversity of D. magna is rich, and the genetic structure of D. magna differs considerably in different regions. These research results and the newly developed polymorphic SSR 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.