Abstract: Triplophysa pappenheimi is a representative species of Nemacheilinae, which can adapt to the aquatic environment of Tibetan Plateau. To investigate the role of hemoglobin gene family in T. pappenheimi adaptation to hypoxia, bioinformatics analysis of hemoglobin gene family was performed based on genome data, followed by an examination of expression changes of hemoglobin genes in response to hypoxia. The hemoglobin gene family to T. pappenheimi consists of 11 members, including six α-globin genes (hbaa1, hbaa2, hbae1, hbae3, hbae4, and hbae5) and five β-globin genes (hbba1, hbba2, hbbe1.1, hbbe1.2, and hbbe2). Motif, domain, and gene structure analysis showed high conservation among these genes; with the exception of hbbe2, the others exhibited similar structure features. Chromosomal localization analysis showed that the members of hemoglobin gene family were distributed on two chromosomes (Chr_07 and Chr_17). The results of physicochemical analysis indicated that all the gene products were stable hydrophobic proteins, except hbbe1.2, among which, hbae3, hbbe1.1, and hbbe2 were classified as acidic proteins, and the other eight were basic proteins, with α-helix is the main secondary structure. Subcellular localization prediction showed that hbae3 and hbae4 were located in the cytoplasm and the other hemoglobin genes were found in the mitochondria. The expression of hbaa1, hbaa2, hbba1 and hbba2 genes in liver, gill and blood showed an up-regulated trend after 12h of acute hypoxia stress, though the difference was not significant (P<0.05). The expression levels of hbaa1, hbba1, hbaa2, and hbba2 genes in liver and blood reached the peak at 24 to 96h under chronic hypoxia (P<0.05). In gill tissue, the highest expression of hbaa1, hbaa2, and hbba1 appeared at 196h after chronic hypoxia, while hbba2 reached its peak at 24h. This study reveals the role of the hemoglobin gene family in hypoxic environment of T. pappenheimi, accumulating valuable scientific data for its conservation.