Abstract: 【Objective】This study aimed to investigate the population structure and diversity differences of fish in the headwater streams of northern and southern foothills of Qinling Mountains， to explore effects of barrages on ecology of fish in upstream and downstream in the headwater streams of northern and southern foothills of Qinling Mountains， so as to provide basic materials for conserving diversity of fish in northern and southern foothills of Qinling Mountains.【Method】Environmental DNA （eDNA） was extracted from water samples of headwater streams of northern and southern foothills of Qinling Mountains. 2×250 bp double-ended sequencing was performed on the Illumina MiSeq 6000 sequen-cing platform. Then the fish diversity was analyzed based on the self-built localized nt database and historical survey data of traditional fish resources.【Result】A total of 94 species of fish， including 11 alien species were detected at 137 sampling sites in the headwater streams of northern and southern foothills of Qinling Mountains belonging to 8 orders， 17 families， and 64 genera. Among them， a total of 91 species belonging to 8 orders， 17 families， and 63 genera were detected in the headwater streams of southern foothill of Qinling Mountains. A total of 79 species belonging to 8 orders， 17 families， and 57 genera were detected in the headwater streams at northern foothill. There were 12 endemic fish species in the headwater streams of southern foothill of Qinling Mountains， including 8 endemic fish species in the headwater streams of Hanjiang River， 3 endemic fish species in the headwater streams of Danjiang River， and Onychostoma fangi endemic to the headwater streams of Jialing River. Only three endemic fish species to the headwater streams of northern foothill. At the level of order， the relative abundance of Cypriniformes was the highest in different headwater streams of the northern and southern foothills， ranging from 74.58% to 78.79%， followed by Siluriformes （7.41%-10.17%）. At the level of genus， the relative abundances of Triplophysa and Rhynchocypris were the highest in the headwater streams of right bank tributaries of Wei River， both accounting for 5.06%. The relative abundance of Rhynchocypris was the highest in the headwater streams of Hanjiang River， accounting for 4.93%. In the headwater streams of Jialing River， the relative abundance of Hemibarbus was the highest， accounting for 5.00%. In the streams of Danjiang River， the relative abundance of Sarcocheilichthys and Carassius were the highest， both accounting for 4.62%. In the 16 streams blocked by a single barrage， the Shannon index and Pielou index of fish species in the upper barrage sections of 14 streams （Shimenzi River and Xun River not included） were lower than those in the lower barrage sections.【Conclusion】Compared with the headwater streams of the northern foothill of Qinling Mountains， the headwater streams of the southern foothill show richer varieties of fish species and more endemic species. The number of alien fish species in the headwater streams of Qinling Mountains exhibits a significant increasing trend， and constructing barrages on the headwater streams disrupts the connectivity of the upstream and downstream river sections， resulting in a decrease in fish diversity.