Chinese sturgeon (Acipenser sinensis) is a large anadromous fish listed under Category I State protectionin China and is critically endangered. This anadromous fish has historical commercial importance and waswidely dispersed in the main tributaries of the Yangtze River and the coastal regions of the Qiantang River, MinjiangRiver, and Pearl River. However, natural populations have declined severely in the last three decades as aresult of blocking of the migration route by the Gezhouba Dam. Natural reproduction of A. sinensis was interruptedduring 2013–2014 according to the monitored number of fish in the breeding population at the GezhoubaDam spawning site and catadromous juvenile fish in the estuary. Fifteen juvenile sturgeons were found in the JiangsuXupu section of the Yangtze River in June 2015. It is of great significance to evaluate whether these juvenilefish are Chinese sturgeon or hybrid sturgeon; however, few studies have tried to identify Chinese sturgeon amongother sturgeons. In this study, we successfully identified and differentiated the newly found juvenile fish fromthree other sturgeon species endemic to China, including 30 A. sinensis, 39 A. dabryanus, and 33 A. schrenckii.Morphological characters, such as bone lamella, gill rakers, and dorsal rays, of these newly found juvenile fishwere consistent with those of Chinese sturgeon. Eleven polymorphic microsatellite loci isolated and characterizedin a previous study were used to identify the three sturgeon species. The results showed that the newly foundsturgeon in the Yangtze River was closest to A. sinensis (Fst=0.039, P<0.05) and farthest from A. schrenckii(Fst=0.209, P<0.05), indicating that these fish were undoubtedly juvenile Chinese sturgeon. The level of geneticdiversity (mean number of alleles=9.455; total heterozygosity=0.796; Shannon-Wiener Diversity Index=1.998) ofthese juvenile Chinese sturgeon was lower than that of other wild Chinese sturgeon (mean number of alleles=13.455; total heterozygosity=0.810; Shannon-Wiener Diversity Index=1.886), suggesting that the size of thewild Chinese sturgeon breeding population is decreasing. No natural A. sinensis reproduction was found during2014 at the Gezhouba Dam spawning site, which is the only known spawning site for this endangered species. Wespeculate that these fish came from another spawning ground. In recent years, the Three Gorges Dam, which is theworld’s largest hydroelectric project, was put on-line, and it has had a tremendous impact on the natural reproductionof Chinese sturgeon by influencing the seasonal fluctuations in downstream water flow and temperatures,potentially resulting in loss of the eco-hydrological conditions needed by the fish for spawning. The Chinese sturgeonis an adaptable fish judging from its life history, so it is likely that other spawning sites located downstreamof the Gezhouba Dam are being used. It is necessary to identify the exact positions of these spawning grounds todescribe the hydrological conditions and better protect this endangered species. Our results will be useful formonitoring natural populations, surveying new spawning sites, and designing conservation strategies.