To determine the basic structural features of the mitochondrial genome, explore the taxonomic status of Diplozoon paradoxum, and to understand the codon usage bias in Diplozoidae species, the mitochondrial genome sequence of D. paradoxum was obtained by the second generation high-throughput sequencing technologies, annotated by MITOS Web Server, and its structure was analyzed by PhyloSuite (version1.2.2). Then, we analyzed the codon composition and usage bias of mitochondrial protein-coding genes in nine species of Diplozoidae. The results showed that the mitochondrial genome sequence of D. paradoxum was 15,713 bp in length (the large non-coding region was not fully sequenced), and the AT content of D. paradoxum was 68.7%, which had an obvious AT bias. Based on the available data, the results of phylogenetic tree analysis showed that all species of Diplozoidae were grouped into one branch, and revealed that Diplozoidae was a monophyletic group. The analysis of codon usage bias showed that the effective number of codons (ENC) ranged from 30.632 to 37.495 in Diplozoidae. The relative synonymous codon usage (RSCU) values of 18 codons were > 1 in Diplozoidae, and there was a bias for codons ending in U(T). The analysis of PR2-plot showed that the frequency of T was higher than that of A, and the frequency of G was higher than that of C. The codon usage bias might be affected by factors such as mutation and selection. The analysis of neutral mapping showed that the effects of mutation pressure on D. paradoxum and P. opsariichthydis were 61.89% and 51.31%, respectively, while the effects of mutation pressure on other seven species were below 50%. Diplozoidae species had slight differences in codon usage bias, but they were all affected by mutation pressure, natural selection, and base composition. This study can provide an important basis for follow-up phylogenetic studies.