Brook trout (Salvelinus fontinalis) belongs to Salmoniformes and Salmonidae. It is native to northeastern North America with fast growth rate and high feed efficiency. Since it was introduced into China, it has become an important cultured species in cold-water. In order to study the population genetic structure and germplasm resource status of the introduced S. fontinalis population, 108 1 pairs of tetranucleotide repetitive microsatellite primers were designed by screening the transcriptome data of S. fontinalis. A total of 200 pairs were selected for primer synthesis, and 111 microsatellite markers with good specificity and high amplification efficiency were obtained through PCR amplification. Twenty-seven pairs of microsatellite primers were selected and used to analyze the polymorphism and genetic diversity of the introduced and cultured populations of S. fontinalis. The results were shown as fellows: a total of 171 alleles were detected in 27 microsatellite markers in two populations. The polymorphism information content (PIC) ranged from 0.426 0 to 0.877 4, with an average of 0.673 1, of which 23 loci were highly polymorphic (PIC ≥ 0.5). The average number of alleles (Na) was 5.555 6 and 4.444 4, the average number of effective alleles (Ne) was 3.914 5 and 3.108 2, the average observed heterozygosity (Ho) was 0.356 2 and 0.265 0, the average expected heterozygosity (He) was 0.700 2 and 0.621 0, and the average PIC was 0.640 9 and 0.555 5, respectively. The genetic parameters of the introduced population were tested by paired sample t test. The results showed that the five genetic diversity parameters of the cultured population were significantly or extremely significantly lower than those of the introduced population, indicating that although the PIC of the cultured population was still at a high polymorphic level (PIC ≥ 0.5), serious allele enrichment had occurred after multiple generations of self-breeding. Using a test of the Hardy-Weinberg principle (χ2 test) and Bonferroni calibration, all except 8 and 4 loci had deviated equilibrium in the introduced and cultured populations respectively, and most loci were heterozygote deficiency. There was high genetic differentiation (Fst=0.164 2), genetic similarity coefficient was 0.582 2 and genetic distance was 0.540 9 between the two populations, which indicated that there was significant genetic differentiation between introduced and cultured populations. These polymorphic markers may also be used in future studies of population genetics, linkage mapping and assisted breeding in S. fontinalis.