The topmouth culter (Culter alburnus) is an economically important fish in China. However, natural stocks have been decreasing rapidly in recent years. Genetic improvement breeding programs of C. alburnus offer an essential strategy in developing a sustainable solution. In this study, we developed three multiplex PCR panels, including twelve microsatellite loci for C. alburnus, and applied parentage analyses using 36 candidate parents and 136 offspring from four independent artificial breeding pilots. Based on allele frequency analysis using 36 candidate parents, the number of alleles ranged from 7 to 22, observed heterozygosity ranged from 0.39 to 0.75, and expected heterozygosity ranged from 0.57 to 0.91. All loci were highly informative (polymorphic information content, PIC >0.5). We detected a significant correlation between exclusion probabilities for parent pairs (E-PP) and PIC (P<0.01). Simulation analysis revealed that a high assignment rate (>95%) was achieved when the number of candidate parents was less than 200. Meanwhile, real parentage analysis revealed that almost each offspring (135/136, 99.26%) was unambiguously assigned to a parent pair with high accuracy (100%), respectively. In addition, we detected significantly unequal progeny contributions of parents and parent pairs in total or in each artificial breeding pilots (P<0.01). In brief, we developed microsatellite multiplex PCR panels for C. alburnus with high capability of monitoring pedigree information, which can be used as a basic tool for family selection breeding of this species.