Objective To analyze the genetic differences between closed colony and wild population of Mugilogobius chulae, and to establish a genetic quality evaluation method of closed colony M. chulae. Methods Twenty microsatellite markers were selected to detect heterozygosity, genetic differentiation, and the relationship between sample size and the genetic parameters of closed colony and wild population of M. chulae. Results The unbiased expected heterozygosity (He) of closed colony and wild population of M. chulae were 0. 6927 and 0. 7162, respectively. Analysis of genetic differentiation showed little difference between closed colony and wild population. Analysis of the relationship between sample size and genetic parameters showed that the mean number of alleles tended to be stable at a sample size of 30. Conclusions Heterozygosity in the range of 0. 5 – 0. 7 can distinguish closed colony strains of M. chulae from wild population, and would be suitable for determining the genetic quality of closed colony M. chulae.