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Amphioxus occupies a transitional position between invertebrates and vertebrates and is therefore a promising laboratory model animal. As a powerful molecular marker in population genetics studies, microsatellite DNA is required for both the investigation of genetic diversity of a wild amphioxus population and strain breeding of laboratory cultured amphioxus. In the present study, we constructed a microsatellite-enriched DNA library of amphioxus Branchiostoma belcheri to develop this molecular marker. After screening the library and combining it with the data obtained from a public database, we successfully developed 21 pair of primers that could effectively amplify the microsatellite loci of amphioxus. Of those primer pairs, seven were in deviation of Hardy–Weinberg equilibrium, mostly caused by null alleles. The others had no linkage disequilibrium, indicating they are applicable for the genetic studies. Using those primer pairs, we further detected the genetic diversity of the wild amphioxus population in Xiamen waters. The results revealed that the number of alleles of each microsatellite locus was between three and 18 in the population, and observed and expected heterozygosities of the loci ranged from 0.415 to 0.947 and from 0.300 to 1.000, indicating very high genetic diversity of the population. [ABSTRACT FROM AUTHOR]