Abstract: Chinese brake fern (Pteris vittata L.) is the most important and well-known arsenic-hyperaccumulating plant used in phytoremediation of arsenic contaminated soils; however, little is known about its genetic diversity. In this study, 100 clones were randomly selected from the library enriched for AG and AC motifs using a modified FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) protocol and sequenced. Fifty-one microsatellite loci, of which 60% were pure repeats, were isolated. Twenty-five pairs of primers were designed and synthesized to evaluate their application and polymorphism in 20 individuals per sampling site obtained from Lushan and Enshi, respectively. Primers of eight loci of pure repeats and one locus of intermittent repeats were finally amplified successfully and yielded clear bands. A total of 41 alleles were detected. The allele number per locus of these microsatellites ranged from two to seven (mean 4.56). The expected heterozygosity (Exp-Het) ranged from 0.0494 to 0.8169. No linkage disequilibrium was found. Cross-species amplification demonstrated that six loci were amplified successfully in P. multifida. The markers helped to reveal the genetic variations of arsenic tolerant genotypes and understand the distribution pattern and ecological adaptation mechanism of P. vittata, and also assisted in breeding new varieties of fern for more effective remediation.