Three Gorges Dam is a vital project in the development and harnessing of the Yangtze River. Water-level fluctuation zone of Three Gorges Reservoir, which refers to the area along the Three Gorges Reservoir banks bounded by minimum (145 m) and maximum (175 m) water levels of the Three Gorges Reservoir, has provoked a series of ecological problems, which included soil erosion, reduction in ecosystem types, decrease in biodiversity and so on. However, effect of water level fluctuating on fish of early life history stages in the water-level fluctuation zone was not yet reported. In this study, the research area was between 150 km and 330 km from Three Gorges Dam, where fish eggs and larvae, exposed to water or remained in shallows, were collected. Based on distribution of sampling places, the research area was divided into 8 regions, which were mainstream (down), Meixi River, mainstream (middle), Shilu River, Modaoxi River, Tangxi River, mainstream (up), and Pengxi River. According to sampling time and place, and morphological characters of fish eggs and larvae, 132 samples were preserved, including 1735 fish eggs and 150 larvae. Twenty-four samples were collected in mainstream. But only one sample in respective mainstream (middle) and mainstream (down) included respective one and nine larvae, in which no fish egg was found. In Modaoxi River, a tributary of Three Gorges Reservoir, there were the most samples in this study, and 59 samples were collected including 733 fish eggs and 108 larvae. Due to insufficient morphological diagnostic characters in kraurotic fish eggs and larvae, it is easy to misidentify in the genus or species level. So, this study was employed DNA barcoding technology to identify the samples. DNA barcoding technology could be used as a rapid tool to distinguish morphologically similar species, and survey many uncertain species and species composition. Previous researches have shown that only molecular identification could guarantee identification of fish eggs and larvae to the species level. Through mtCO I sequence alignment, and based on mtCO I sequences similarity values (species:>99%, genus:92%-99%, family:85%-92%), the samples were distinguished to 14 types, of which 10 types were identified to species:Cyprinuscarpio, Carassiusauratus, Hemiculterleucisculus, Rhinogobius nagoyae, Rhinogobius giurinus, Opsariichthysbidens, Acheilognathusmacro-pterus, Pseudorasboraparva, Hypophthalmichthys molitrix, and Rhinogobius, Rhodeus, and Rhodeusocellatus and . If fish DNA barcode database of Yangtze River or Three Gorges Reservoir could be completely and accurately constructed, and new molecular markers could be developed for fishes that can be not distinguished by mtCO I barcode, it would help to further identify fish eggs and larvae in the water-level fluctuation zone and investigate fish resources of the Yangtze River. had the largest proportion in the samples, 50.0% and 24.6% respectively, and widely distributed. The number and species of samples in upstream of mainstream was more than downstream in the research area. It may be not only related to its topography, and also its fish resources. In Modaoxi River, the number and species of samples was the most in this research area, which contained nine fish species. The result showed that it was more affected from the water level fluctuating, at the same time, it also indicated that fish resource were rich in Modaoxi River, which should be got more attention in the future fish resources investigation and protection in Three Gorges Reservoir. This study employed DNA barcoding technology to identify fish eggs and larvae collected in partial water-level fluctuation zone, which would contribute to comprehensively assessing the effect of water level fluctuating on fish resources in water-level fluctuation zone of Three Gorges Reservoir.