As aquaculture food production is expected to increase, introduced aquaculture species play an important role in meeting the rising demand for aquatic food products and contributing to great societal benefits. Species introduction forces sympatric species to coexist within the same ecosystem by niche segregation. Japanese smelt Hypomesus nipponensis and sharpbelly Hemiculter leucisculus are ecologically similar species and were introduced to Lake Bosten, the largest inland lake in Northwest China, accounting for more than 60% of the total production. We predicted that the coexistence of the two invasive species is mediated by habitat segregation. We analyzed spatiotemporal patterns of Japanese smelt and sharpbelly abundance in Lake Bosten to determine the patterns of spatial segregation between the two dominant fish species. Our results showed that, in Lake Bosten, sharpbelly are typically littoral dwellers when the surface temperature increases in spring and summer, while Japanese smelt often prefer pelagic waters, especially during summer and autumn when the surface temperature rises. Japanese smelt showed an affinity for deeper waters, using median depths 1.7 m deeper than those of sharpbelly, irrespective of the sampling season. Water temperature was the main underlying driver of such spatial segregation and coexistence of these two closely related species in Lake Bosten. Spawning, food resources, and fishing were also important possible factors affecting spatial segregation between the two species. Our results provide new information on niche partitioning patterns as strategies for the coexistence of the two dominant non-native fish in Lake Bosten, supporting the idea that ecologically similar species can avoid resource competition through spatial habitat segregation.