Branchiostoma belcheri Gray is an endangered species. A set of twelve novel polymorphic microsatellite loci was developed in B. belcheri Gray. The polymorphism of these loci was tested on a population of thirty individuals from Xiamen, China. The number of alleles ranged from 3 to 7. The polymorphism information content ranged from 0.309 to 0.778. The levels of expected and observed heterozygosities varied from 0.2449 to 0.7825, and from 0.1786 to 0.8571, respectively. Three loci (WCY4-22, WCY4-39, WCY5-17) deviated from Hardy–Weinberg equilibrium. These microsatellite loci would contribute to population genetic studies and conservation of B. belcheri Gray.