Abstract Background The schizothoracine fishes, an excellent model for several studies, is a dominant fish group of the Qinghai-Tibet Plateau (QTP). However, species populations have rapidly declined due to various factors, and infection with Echinorhynchus gymnocyprii is cited as a possible factor. In the present study, the molecular characteristics of E. gymnocyprii in four species of schizothoracine fishes from the QTP were explored. Methods We investigated the infection status of E. gymnocyprii in 156 schizothoracine fishes from the upper Yangtze River, upper Yellow River, and Qinghai Lake in Qinghai Province, China. The complete internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) gene and part of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of 35 E. gymnocyprii isolates from these fishes were sequenced and their characteristics analyzed. In addition, we inferred phylogenetic relationships of the E. gymnocyprii populations based on the rRNA-ITS and cox1 sequences. Results The total prevalence of E. gymnocyprii in schizothoracine fishes was 57.69% (90/156). However, the prevalence among different species as well as that across the geographical locations of the schizothoracine fishes was significantly different. The results of sequence analysis showed that the four E. gymnocyprii populations from different hosts and regions of Qinghai Province were conspecific, exhibiting rich genetic diversity. Phylogenetic analysis based on rRNA-ITS and cox1 sequences supported the coalescence of branches within E. gymnocyprii; the cox1 gene of E. gymnocyprii populations inferred some geographical associations with water systems. In addition, three species of schizothoracine fishes were recorded as new definitive hosts for E. gymnocyprii. Conclusions To the best of our knowledge, this is the first molecular description of E. gymnocyprii populations in schizothoracine fishes from the Qinghai-Tibet Plateau that provides basic data for epidemiological surveillance and control of acanthocephaliasis to protect endemic fish stocks.