In this study, we successfully assembled the complete mitochondrial genome of the Amu Darya sturgeon Pseudoscaphirhynchus kaufmanni. Based on this mitochondrial genome and previously published mitochondrial genomes of members of the Acipenseridae family, we assessed the phylogenetic position of P. kaufmanni using maximum likelihood and Bayesian inference for phylogeny reconstruction. The resultant phylogenetic trees were well-resolved, with congruence between different phylogenetic methods. This robust phylogenetic analysis elucidated the relationship among the four acipenserid genera and strongly supported the division of the family into three main clades. Evaluation of molecular phylogeny using maximum likelihood and Bayesian analysis led to the following conclusions: (a) the most basal position within the Acipenseridae remains in the clade containing Acipenser oxyrinchus and Acipenser sturio; (b) the genus Scaphirhynchus belongs to the Atlantic clade and is a sister group of the remaining species of the clade; and (c) the close relationship between P. kaufmanni and Acipenser stellatus is well supported.