The closure of the Isthmus of Panama (about 3.1 million years ago) separated previously continuous populations and created two groups of extant species, which live now in the Pacific and Atlantic drainage systems. This relatively recent event was a trigger to diversification of various species in the Neotropics, nonetheless there are exemplars that do not show sufficient morphologic variability to separate them by traditional morphological tools. About 60 years ago, some freshwater decapod species with high morphological similarity were separate by previous researchers, based on geographical distribution, in Pacific and Atlantic and considered as “sister species”. However, the complete isolation of these prawns by this geographical barrier is questionable, and it has generated doubts about the status of the following transisthmian pairs of sibling species: Macrobrachium occidentale × Macrobrachium heterochirus, Macrobrachium americanum × Macrobrachium carcinus, Macrobrachium digueti × Macrobrachium olfersii, Macrobrachium hancocki × Macrobrachium crenulatum, Macrobrachium tenellum × Macrobrachium acanthurus and Macrobrachium panamense × Macrobrachium amazonicum. Here we evaluated the relation among these pairs of sibling species in a molecular phylogenetic context. We generated 95 new sequences: 26 sequences of 16S rDNA, 25 of COI mtDNA and 44 of 18S nDNA. In total, 181 sequences were analyzed by maximum likelihood phylogenetic method, including 12 Macrobrachium transisthmian species, as well as seven other American Macrobrachium species, and two other palaemonids. Our analysis corroborated the morphological proximity of the sibling species. Despite the high degree of morphological similarities and considerable genetic diversification encountered among the transisthmian sister species, our data support the conclusion that all species included in sibling groups studied herein are valid taxonomic entities, but not all pairs of siblings form natural groups.