The organization of fish mitochondrial genomes (mitogenomes) is quite conserved, usually with the heavy strand encoding 12 of 13 protein-coding genes and 14 of 22 tRNA genes, and the light strand encoding ND6 and the remaining 8 tRNA genes. Currently, there arc only a few reports on gene reorganization of fish mitogenomes, with only two types of rearrangements (shuffling and translocation) observed. No gene inversion has been detected in approximately 420 complete fish mitogenomes available so far. Here we report a novel rearrangement in the mitogenome of Cynoglossus semilaevis (Cynoglossinae, Cynoglossidae, Pleuronectiformes). The genome is 16371 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 main noncoding regions, the putative control region and the light-strand replication origin. A striking finding of this study is that the tRNA(Gln) gene is translocated from the light to the heavy strand (Q inversion). This is accompanied by shuffling of the tRNA(Ile) gene and long-range translocation of the putative control region downstream to a site between NDI and the tRNA(Gln) gene. The remaining gene order is identical to that of typical Fish mitogenomes. Additionally, unique characters of this mitogenome, including a high A+T content and length variations of 8 protein-coding genes, were found through comparison of the mitogenome sequence with those from other flatfishes. All the features detected and their relationships with the rearrangements, as well as a possible rearrangement pathway, are discussed. These data provide interesting information for better understanding the molecular mechanisms of gene reorganization in fish mitogenomes.