A recent taxonomic revision indicated that Macrobrachiumpotiuna, an endemic prawn in Brazilian freshwater drainages, exhibits wide morphological variability along its limited geographical distribution. However, in some cases, taxonomic doubts at the species level have no clear morphological resolution. Considering that no molecular data of M. potiuna along its distribution were available to provide a complete and integrated overview, we analyzed 21 partial sequences (531 bp) from the mitochondrial 16S rRNA gene of M. potiuna and 9 sequences from outgroup species, by maximum likelihood and parsimony, in order to investigate the possibility of the existence of cryptic species, within the morphologically based M. potiuna. The topologies obtained revealed that M. potiuna represents a monophyletic clade. Nevertheless, two clades supported by both analyses were formed within the M. potiuna taxon. The mean genetic divergence between these two groups was 0.044 ± 0.007, and within each group (i.e., M. potiuna "sensu stricto" andM. potiuna "Affinis-Clade") the divergences were 0.010 ± 0.003 and 0.028 ± 0.005, respectively. As far as we know, this is the first report to show a genetic separation between populations of prawns with abbreviated larval development in South American drainages. Pending additional analysis, to propose a conclusive inference, the existence of these distinct genetic groups must be considered in future studies with the morphologically based M. potiuna. In addition, we extended the known northern distribution with a record from the state of Bahia.