Abstract: In this study, six complete mitochondrial genomes of Acipenseriforemes (Acipenser sinensis, A. dabryanus, A. brevirostrum, A. naccarii, Huso dauricus and Polyodon spathula) were successfully assembled. Based on 6 novel mitogenomes and 17 previously published mitogenomes of sturgeons and paddlefishes, the molecular phylogeny of Acipenseriformes was constructed by maximum likelihood and Bayesian method, and the tree topologies were evaluated by likelihoods value test. The results showed that the length of entire mitochondrial genomes of the six species were 16521—16766 bp, encoding 13 protein-coding genes, 22 tRNA genes and two rRNA gene, which were similar to those of other sturgeons and paddlefishes. Molecular phylogenetic analyses showed that (i) the Acipenseriformes contained two monophyly families (Polyodontidae and Acipenseridae); (ii) genus Huso and Acipenser were demonstrated as non-monophyletic and the family Acipenseridae can be divided into 3 subgroups: A. sturio-A. oxyrinchus clade, Atlantic clade and Pacific clade. Tree topology tests indicated that the phylogenetic relationship of the subfamily Acipenseridae is A. sturio-A. oxyrinchus clade (Pacific clade, Atlantic clade). The species of genus Scaphirhynchus has occupied the basal position of the Atlantic clade in Acipenseridae. This study showed that mitochondrial genome data has important application value in the systematic and evolutionary study of Acipenseriformes.