Abstract: In order to solve the problems in paternity test and genealogical management for improved variety breeding of Monopterus albus, we obtained 16 microsatellite markers with high polymorphism through whole genome searching of and following confirmation, and established two multiplex PCR sets by using 16 high polymorphic microsatellite markers and successfully applied to the parentage assignment for 11 full-sib M. albus families. The average allele number(N_a) of 16 microsatellite was 5.562; the average of abserved heterozygosite(H_o) was 0.627; the average of expected heterozygosite(H_e) was 0.619; the average of polymorphic information content (PIC) was 0.564. Parentage analysis reveals that the combined exclusion probability of first parent given only the genotype of the offspring (CE-1P) was 0.999 999 99, the combined exclusion probability of a second candidate parent given the genotype of the offspring and one known parent (CE-2P) was 0.999 999 91, and combined exclusion probability of a parent pair given only the genotype of the offspring (CE-PP) was 0.999 964 76. The simulated identification rate of the 11 M. albus families was 99.96%, and the actual identification rate was 95%. Furthermore, the simulation analysis shows that the identification rate could reach over 95% in the case of 200 candidate parental individuals with known sexes and 150 candidate parental individuals with unknown sexes. The cluster analysis shows that 108 offspring could be clustered correctly except two, and the accuracy rate was 98.18%. The paternity method of M. albus established in this study provides important technical support for the future breeding program and management of germplasm resources of M. albus.