Saline-alkaline water use has broadened the development of Chinese fisheries. Breeding new varieties ofalkaline-resistant fish is a fundamental task, and elucidating fish alkaline-resistant mechanisms, identifying candidategenes, and exploring their functions are crucial. Decoding genome information promotes the use of molecular markersand selection of candidate genes. Leuciscus waleckii (Dybowski) is highly tolerant to high salinity and alkalinity andsurvives in Dali Lake (Inner Mongolia) with alkalinity up to 53.57 mmol/L (pH 9.6). Therefore, this fish species is agood subject to investigate the evolutionary mechanism of high salinity and alkalinity adaptation. L. waleckii in DaliLake differ from their freshwater counterparts because they are characterized by a breeding migration in which theyspawn in freshwater and grow in alkaline water. Marked differences are observed during acute-phase alkaline tolerancebetween the alkaline population from Dali Lake and the freshwater population from the Songhuajiang River, indicatingthe genetic heritability of alkaline tolerance. However, the mechanism of high tolerance to alkalinity remains unknown,and very few physiological and genetic studies have been performed. Thus, we prepared a hybrid F2 system (alkalineDali Leuciscus × freshwater Songhua Leuciscus). A large number of candidate genes are associated with alkaline-resistance traits, and microsatellites, single nucleotide polymorphisms, and other molecular markers have been appliedin fish population genetics studies. We used the gene recombination principle to select molecular markers closelyrelated to alkaline-resistance traits and located functional genes using molecular markers. In this study, we examinedand genotyped alkaline tolerance in 77 hybrid F2 individuals using 39 simple sequence repeat (SSR) markers, including19 expressed sequence tag EST-SSRs. The individuals were divided into three groups of 43, 20, and 14 individualsbased on their alkaline tolerance. Fisher’s exact probability test was used to examine the associations between markersand the trait. The results showed that two markers were strongly linked to alkaline tolerance (P<0.05), and one was anEST-SSR. Sequence alignment of that EST-SSR showed that it was highly homologous with the hypoxia inducible factorHIF-3 gene of Wuchang bream (Megalobrama amblycephala) and the HIF-4 gene of grass carp (Ctenopharyngodonidellus). These genes are involved in the response to hypoxia and increased hypoxia tolerance in fish. The approachwas to select a functional gene using molecular markers, localize the functional gene, and narrow the range ofcandidate genes to lay the foundation for breeding new varieties of alkaline-resistant fish. In-depth study of this functionalgene will be conducted in the future. Our results suggest further analyses of candidate alkaline-responsive genes,which will help in the understanding of teleost adaptation under extreme environmental stress and ultimately benefitfuture breeding for an alkaline-tolerant fish strain.