Schizothorax prenanti (S. prenanti), one of the important endemic commercial fish in China, is mainly distributing in the upstream of the River Yangtze and the tributaries. The wild population is facing serious challenges of germplasm degeneration due to the overfishing, water pollution and construction of hydropower stations; therefore, it is very urgent to develop genetic resource of S. prenanti to protect the wild population. In this study, we used Illumina Hiseq2500 sequencing to develop single nucleotide polymorphism (SNP) markers in S. prenanti. From 37,785 unigenes with functional annotation, 857,535 putative SNPs were identified. Among them, 33 SNPs from immune-related genes were randomly selected and 20 loci exhibited significant polymorphisms in genotyping by Sequenom MassARRAY. As far as our best knowledge, this is the first report about the SNP markers development in S. prenanti based on Illumina RNA sequencing. These SNP markers should not only be useful for population conversation, but also for construction of genetic linkage map and economic performance improvement of S. prenanti.