Abstract: Using magnetic bead enrichment, microsatellite sequences of Sphaeropteris brunoniana were collected. Through primers designed and screened, polymorphism analysis and validation were conducted on the developed primers. Further genetic diversity assessment was conducted, aiming to develop efficient molecular markers for germplasm resource studies in S. brunoniana. The results showed that 40 pairs of primer sequences were successfully designed, including 29 pairs of perfect repeat sequences(72.5%), 9 pairs of non-perfect(22.5%), and 2 pair of composite(5%). The 23 pairs of polymorphic primer pairs were screened, with 149 polymorphic bands, 96.13% polymorphic band ratio, average number of observed alleles(Na) of 1.878, average number of effective alleles(Ne) of 1.422, average value of Nei's gene diversity index(H) of 0.272, average value of Shannon's information index(I) with a mean value of 0.423, expected heterozygosity(He) with a mean value of 0.272, and polymorphism information content(PIC) ranging from 0.152-0.917 with a mean value of 0.671. Based on the SSR markers, the participating materials can be classified into two populations according to their geographical origin. The SSR primers developed this time have high availability and provide new molecular marker tools for future work such as assessing genetic diversity and molecular breeding of S. brunoniana.