We report the first mitochondrial genomes of the Afrobatrachian frog species Hyperolius substriatus, providing insights into the gene arrangements, duplications, and evolutionary dynamics within this taxonomically complex genus. Assemblies were gained from two museum specimens and included all 37 typical vertebrate mitogenomic genes. In both cases, the mitogenome of H. substriatus shows duplications of tRNAs (trnL, trnT and trnP). The WANCY region exhibits the characteristic Afrobatrachian pattern but with unique intergenic spacers. Both copies of duplicated non-coding and control regions show high sequence similarity within and between specimens. The distinct gene order and homology of duplications observed can be explained under the tandem duplication and random loss model of mitochondrial evolution. Furthermore, a comparison with Hyperolius marmoratus highlights differences in gene copy number and synteny, and the presence of a duplication of trnM in the latter appears to be a derived condition. Together, these findings provide valuable insights into the taxonomy and evolution of Hyperolius, contributing to understanding interspecific gene reorganisation in this diverse group and suggesting that gene arrangements could help resolve some of the taxonomic problems of this complex genus.