Hypoptopoma incognitum is a fish of the fifth most species-rich family of vertebrates and abundant in rivers from the Brazilian Amazon. Only two species of Loricariidae fish have their complete mitogenomes sequence deposited in the Genbank. An innovative RNA-based approach was used to assemble the complete mitogenome of H. incognitum with an average coverage depth of 5292×. The typical vertebrate mitochondrial features were found; 22 tRNA genes, two rRNA genes, 13 protein-coding genes, and a non-coding control region. Moreover, the use of this approach allowed the measurement of mtRNA expression levels, the punctuation pattern of editing, and the detection of heteroplasmies.