Gymnocypris przewalskii is an important indigenous economic fish in Qinghai Lake, which plays a key role in the whole Qinghai Lake ecosystem and shows a strong saline-alkali tolerance and adaptability compared with other fishes. In order to explore the important functional genes of G. przewalskii during the process of saline-alkali tolerance, Illumina Hiseq technology was used to define the transcriptome of gill and kidney tissues of G. przewalskii in two areas (Qinghai Lake and the Quanji River). All kinds of clean data reached more than 6.74 Gb, and the percentage of q30 base was more than 94.38%. After quality control and assembly, a total of 541 429 unigenes were obtained, the average length of N50 was 612 bp. The sequences acquired were blasted against NR, Swiss-Prot, pfam, COG, GO and KEGG databases, finally, 107 568 unigenes could be annotated. Differential expression analysis showed that a total of 832 genes were co-expressed in the gills and kidneys in two regions. Gene ontology analysis indicated that differentially expressed genes (DEGs) annotated to binding, cellular process, metabolic process, and single-organism process accounted for a large proportion. Based on KEGG pathway analysis, the pathways related to immunity, metabolism and penetration were enriched. According to the GO annotation of DEGs and the enrichment analysis of KEGG signaling pathway, we initially screened 35, 34, and 25 genes related to osmotic, immunity, and metabolism of G. przewalskii, respectively. The osmotic related genes included calcium/calmodulin-dependent protein kinase, sodium/potassium transporting ATPase, and mitogen-activated protein kinase, solute carrier family, etc, immune related genes mainly included interleukin, complement, integrin, granzyme B, etc; nitric oxide synthase, 1,25-dihydroxyvitamin D (3) 24-hydroxylase,cytochrome P450 and elongation factor were related to metabolism of G. przewalskii. Sixteen differentially DEGs were randomly selected for quantitative real-time PCR (qRT-PCR), and the results were consistent with the RNA-seq. The result of this study enriches the basic data of G. przewalskii’s omics research, and also provides a reference for further study of G. przewalskii in saline-alkali tolerance.