The next-generation sequencing technology is obviously faster than Sanger sequencing technology, and the cost is basically the same. Based on this, we used the next-generation sequencing technology to measure the whole mitochondrial genome sequence of Ammodytes personatus, and compared with Sanger sequencing results. Three samples belonging to two lineages of A. personatus mitochondrial DNA (mtDNA) were used in the study. Based on the complete mtDNA sequence, the genetic distance between the two lineages is 0.064. Results show that the total sequence lengths of the three samples of A. personatus mitochondrial genome are 16,537 bp (AP1), 16,536 bp (AP2) and 16,537 bp (AP3), where the base composition has biases of high A+T and low G+C contents, which are typical to the structural composition of vertebrates. Moreover, the two promoters (ATG, GTG) and four terminators (TAG, AGG, TAA, T or TA) in the 13 protein-coding genes are found. Except for tRNA-Ser(AGN), the second-structure of other tRNAs is the typical clover structure. The lengths of 12S rRNA and 16S rRNA are 945 and 1,698 bp (AP1, AP2) and 1,696 (AP3). A control region containing key sequence tags have three different domains, namely, terminating sequences (TAS1, TAS2), central conservatives (CSB-F, CSB-E and CSB-D) and conservative sequences (CSB1, CSB2 and CSB3). A conserved sequence region that controls the initiation of light-chain replication is identified in the non-coding region outside the control area. Based on the complete mitochondrial genome and COI gene, we can identify the phylogenetic relationship of A. personatus to other Perciformes species. Our results clearly demonstrate that the high-throughput sequencing method is the methodology of choice for generating complete mtDNA genome sequences.