Abstract: During stock enhancement activities, to effectively protect the genetic diversity of amphioxus germplasm resources, this study employed microsatellite marker technology to conduct a comparative analysis of the genetic diversity and genetic structure between wild and released amphioxus populations. Primers were designed based on microsatellite sequences obtained from amphioxus transcriptome sequencing. Eight polymorphic microsatellite loci were selected, and capillary electrophoresis was employed to assess the genetic diversity of wild and released populations（2023−2024）. A total of 122 alleles were amplified across the eight loci, with the number of alleles per locus ranging from 7 to 25（average: 15.15）. The polymorphism information content（0.616−0.930）indicated high genetic polymorphism. The average expected heterozygosity of wild and released populations was 0.762−0.817, and the average allelic richness was 9.631−11.382. The genetic distance between wild and released populations was relatively small（0.015−0.045）, suggesting low genetic differentiation. Both wild and released populations of amphioxus exhibit high genetic diversity, with the wild population showing slightly higher diversity. However, no significant genetic structure differentiation was observed between the two groups. This study provides a fundamental genetic basis for the restocking and germplasm resource conservation of amphioxus.