Abstract: Rhynchochypris lagowskii is a small freshwater economic fish species selected by the Ministry of Agriculture and Rural Affairs as one of the top 10 characteristic aquatic germplasm resources in 2022. To investigate the genetic diversity of the R. lagowskii populations in northern China and provide references for its conservation and genetic breeding, Genotyping by sequencing (GBS) was used to develop single nucleotide polymorphism (SNP) markers and analyze genetic characteristics of 109 individuals from four R. lagowskii populations in Ninghe, Jizhou, Juhe (Tianjin), and Taizihe (Liaoning). Through sequencing, a total of 3723530 SNP markers were obtained, and 133336high-quality SNP loci were screened for genetic analysis after filtering. The expected heterozygosity (He) of the four populations was 0.0397—0.0884, the observed heterozygosity (Ho) was 0.0394—0.0818, the nucleotide diversity (Pi) was 0.0406—0.098, the polymorphic information content (PIC) value was 0.0327—0.0717, the genetic differentiation coefficient (F_ST) was 0.0039—0.8148, and the inbreeding coefficient (Fis) within population was 0.0567—0.5480. Genetic structure analysis showed that four populations clustered into three distinct groups, with individuals from the Ninghe and Jizhou populations in Tianjin clustered into one branch, and individuals from the Taizihe and Juhe populations in Liaoning and Tianjin clustered into one branch, respectively. Population specific SNP markers were identified for all four populations, serving as candidate molecular markers for species identification, population genetics, trait association analysis, and evolutionary studies. Population history dynamics research indicates that the four populations of R. lagowskii all underwent a process characterized by a sharp decline in effective population size (N_e) during the Quaternary glacial period.