The genus Neacomys includes 10 recognized species of Neotropical spiny mice in the tribe Oryzomyini. Five species have previously been reported from Peru, but the small-bodied Peruvian taxa remain unrevised. In this report, we present the first systematic and taxonomic revision of small-bodied Neacomys populations in Peru and describe two new species based on molecular, morphological, and karyotype data: (1) Neacomys rosalindae, sp. nov., from northeastern Peru, is distinguished from congeneric species by, among other differences, short incisive foramina with a wide maxillary portion of the septum, a small subsquamosal fenestra, and a karyotype of 2n = 48, FN = 50. (2) Neacomys macedoruizi, sp. nov., from central Peru, is distinguished by its gray-based ventral fur, large infraorbital foramen, and karyotype of 2n = 28, FN = 36, with a distinctively large pair of metacentric chromosomes. The results of our molecular analyses suggest that N. minutus (as currently recognized) is a species complex comprised of N. minutus sensu stricto, N. macedoruizi, and a third form that remains to be described. The other species described here, N. rosalindae, is the sister taxon to a cluster that includes the N. minutus complex plus N. musseri. Our data suggest that the upper Amazon River constitutes an important dispersal barrier for species in this genus.