One of the last wild populations of the critically endangered stellate sturgeon (Acipenser stellatus) survives in the Danube River. Limited knowledge about the genetic structure, ecology, and evolution of this species led to poor and inconsistent management decisions with an increased risk for species extinction in the wild. Here we show the results of genetic structure screening of the Danube River wild population over 12 years timespan. Our research does not bring evidence of population recovery. No genetic structuring was identified at the mitochondrial level concerning spawning migration timing, sampling locations, and developmental stages. Eleven maternal lineages were revealed based on restriction fragment lengths analysis of the D-loop region, with one haplotype as the most frequent. While this could be the result of a massive restocking activity using a reduced number of spawners, our data does not support it. The selection of mitochondrial haplotypes under the pressure of habitat contraction and the narrower range of temperature variation since dams’ construction on the river could explain the observed distribution. Several factors of managerial concern are discussed. Our results provide baseline data on the mtDNA diversity in a critically endangered species of exceptionally high socioeconomic and conservation interest.