BACKGROUND: Macrobrachium amazonicum is a freshwater prawn widely distributed in South America that is undergoing speciation, so the denomination "M. amazonicum complex" is used for it. The mitochondrial cytochrome c oxidase subunit I (COI) gene has been used to elucidate this speciation, but heteroplasmies and pseudogenes have been recorded, making separation difficult. Obtaining genes from cDNA (RNA) rather than genomic DNA is an effective tool to mitigate those two types of occurrences. The aim of this study was to assemble in silico the mitochondrial DNA (mtDNA) of the Amazonian coastal population of M. amazonicum inhabiting the state of Pará. RESULTS: Sequences were obtained from the prawn's transcriptome using the de novo approach. Six libraries of cDNA from the androgen gland, hepatopancreas, and muscle tissue were used. The mtDNA of M. amazonicum was 14,960 bp in length. It contained 13 protein-coding genes, 21 complete transfer RNAs, and the 12S and 16S subunits of ribosomal RNA. All regions were found on the light strand except tRNAGln, which was on the heavy strand. The control region (D-loop) was not recovered, making for a gap of 793 bp. The cladogram showed the formation of the well-defined Macrobrachium clade, with high support value in the established branches (91-100). The three-dimensional spatial conformation of the mtDNA-encoded proteins showed that most of them were mainly composed of major α-helices that typically shows in those proteins inserted in the membrane (mitochondrial). CONCLUSIONS: It was possible to assemble a large part of the mitochondrial genome of M. amazonicum in silico using data from other genomes deposited in GenBank and to validate it through the similarities between its COI and 16S genes and those from animals of the same region deposited in GenBank. Depositing the M. amazonicum mtDNA sequences in GenBank may help solve the taxonomic problems recorded for the species, in addition to providing complete sequences of candidate coding genes for use as biomarkers in ecological studies.