This study aimed to investigate the genetic structure and variability within the clearhead icefish (Protosalanx chinensis) population by developing 18 microsatellite (simple repeat sequence, SSR) primers for population genetic analyses. In total, 281 samples of P. chinensis were collected from four water systems and eight water bodies since 2016. The results were as follows: 172 alleles were detected, with an average of 9.6 alleles (N_a) ranging from 3 to 24; the number of effective alleles (N_e) ranged from 1.039 to 4.595, with an average of 2.384; the expected heterozygosity (H_e) ranged from 0.035 to 0.804, with an average value of 0.507; and the polymorphic information content (PIC) ranged from 0.034 to 0.775, with an average of 0.469. Notably, 10 of these loci exhibited high polymorphism with PIC values exceeding 0.5. Regarding population-level statistics, the average allele number (N_a) of the eight water populations was 3.389–5.389 and the average value was 4.375. The polymorphic information content (PIC) was 0.373–0.479, with an average value of 0.419. Analysis of Molecular Variance (AMOVA) showed that 84.54% of the total variation in P. chinensis came from within populations and 15.46% from genetic variation among populations. Additionally, a Principal Component Analysis (PCA) was conducted on eight geographic populations of P. chinensis, and all individuals were divided into three subgroups. Nei's genetic distance was employed to construct a UPGMA cluster tree, which classified the eight water bodies into two major branches. Among these, the five water groups of Lianhuan Lake displayed considerable diversity, with Nashidai (NS) and Talahong (TL) forming clusters that subsequently merged with Erbaguzi (EB). Similarly, Dalonghu (DL) and Amuta (AM) grouped, indicating that these five water bodies constituted independent entities within the Lianhuan Lake. Moreover, Xingkai Lake (XK) and Xinlicheng Reservoir (XLC) were grouped, followed by their combination with Taihu Lake (TH) as another unit. Research has shown that the northern population of P. chinensis maintains a high level of genetic diversity and exhibits significant genetic differentiation from populations in different water bodies. To safeguard and preserve population diversity, it is imperative to recognize and protect the distinct statuses of both the southern and northern water system populations. In particular, the preservation of Lianhuan Lake, which serves as the primary source of P. chinensis, is of paramount importance.