Species of Neacomys are small cricetid rodents that occur in forested habitats of Central and South America, from eastern Panama to central Bolivia and central/western Brazil. In order to assess species diversity of this poorly known genus, we obtained cytochrome b gene sequences from the most comprehensive taxonomic and geographic sampling analyzed to date. We also conducted morphological analyses on a large series of specimens housed in 15 museums, including types of 10 out of 14 nominal taxa. Our analyses of the genetic data recovered 17 lineages clustered in four distinct clades. Among these lineages, 11 correspond to species currently recognized as valid, and the remaining six are putative new species. In southeastern Amazonia—the geographical scope of this report—four undescribed species were discovered, three of which are named herein: Neacomys marajoara, sp. nov., from the Island of Marajó, Pará state; Neacomys vossi, sp. nov., restricted to the Tapajós center of endemism (between the Tapajós and Xingu rivers); and Neacomys xingu, sp. nov., restricted to the Xingu center of endemism (between the Xingu and Araguaia/Tocantins rivers). The new species can be discriminated from other Neacomys species by the morphology of the nasal bones, zygomatic plate, interorbital region, subsquamosal fenestra, paraoccipital process, incisive foramina, auditory bullae, anterocone and anteroloph of the first upper molar, carotid circulation pattern, and karyotype. Our results substantially improve our understanding of the genus Neacomys by providing morphological, morphometric, and novel molecular insights about these poorly known rodents and demonstrate that the diversity of small Amazonian mammals is still poorly known, even in the relatively accessible southeastern part of the biome.