Abstract: Silurus lanzhouensis, the name card of the aquatic organism of the Yellow River, is an ecological resource of important value. As a key protected commercial fish in the Upper and Middle Yellow River, S. lanzhouensis has a broad breeding prospect. For the ecological civilization construction of the Yellow River, strengthening the rescue practices, breeding strategies, and scientific utilization of the S. lanzhouensis germplasm resources are of great significance. Aiming to figure out problems of paternity identification and genealogy management, to rescue and preserve S. lanzhouensis germplasm resources and thus to improve variety selection, two sets of quadruple PCR and two sets of triple PCR systems have been established using fluorescent primers and automatic sequencing technology, which have been successfully applied to the paternity test in three families. Performing the analysis of the genetic diversity of 110 fish of S. lanzhouensis using Cervus v.3.0 software, the results obtained from the 14 microsatellite markers screened in this study revealed the average of observed heterozygosity (H_o) 0.750, the average of expected heterozygosity (H_e) 0.667, and the average of polymorphic information content (PIC) 0.624, indicating rich igenetic diversity. The parentage assignment was performed on 90 offspring and 20 candidate parents from three S. lanzhouensis family with known pedigree information. The results showed that the combined exclusion probabilities of the first parent (CE-1P), the second parent (CE-2P), and a parent pair (CE-PP) 0.99753092, 0.99983971, and 0.99999964, respectively. The cumulative simulated identification rate of the four sets of multiplex PCR was 100%, and the cumulative actual identification rate was 83%. Double-blind validation was performed on 50 offspring from 3 families, and cluster analysis was performed using MEGA7.0. The result that the cluster analysis results of 94% of the individuals from the same family was consistent with the genealogical relationship. It is concluded that the four sets of technical system for paternity test using multiplex PCR can provide important technical supports for the polyculture of S. lanzhouensis with different germplasm, germplasm selection, pedigree management and molecular marker-assisted selection.