In China, the brown trout is a second-level key aquatic wildlife protection animal in the Tibet Autonomous Region. In order to protect the brown trout germplasm resources in the Tibet Autonomous Region, the Institute of Aquatic Sciences of the Academy of Agricultural and Animal Husbandry Sciences of the Tibet Autonomous Region and the Heilongjiang Institute of the Chinese Academy of Aquatic Sciences collaborated on research on the key technologies of brown trout family construction. A fast in silico simulation method was employed in this study to discover numerous polymorphic SSRs. A total of 1,115,563 SSR loci with two to six nucleotide repeat motifs were mined from 40 chromosomes of the brown trout reference genome by GMATA. The whole genome sequences (30×) of 30 brown trout samples were compared to the reference genome’s chromosome sequence. Then, the HipSTR program was employed to carry out in silico genotyping through comparisons and enumerations of repeat count variants on the motifs of the SSR loci among the samples. The loci with monomorphism and high deletion in the SSR genotype data were removed, and the loci with more than one non-reference allele were retained. The final set of polymorphic SSR loci screened was 106,194. Forty-three SSR markers were randomly selected for characteristic analysis. The range of alleles per locus was 3 to 9 (mean = 5). Observed heterozygosity was in the ranged from 0.167 to 1.000. Expected heterozygosity was in the ranged from 0.155 to 0.823. Twenty-three loci significantly deviated from Hardy-Weinberg equilibrium. These SSR markers will provide valuable information for assessing the genetic diversity of constructed brown trout families and for the genetic improvement of brown trout.