Phylogenetic relationships among available samples of the genus Isothrix using mitocondrial gene cytochrome b were carried out and a new karyotype of Isothrix specimens referred to Isothrix negrensis from the mid course of Rio Negro, in the Brazilian Amazon, was described. Molecular, karyological and morphological data of Isothrix negrensis was compared to that of I. bistriata and I. pagurus. Cytochrome b DNA sequence analyses clearly separate I. negrensis from I. bistriata and genetic distances estimates between then are greater than between species of the related genus Mesomys. The median-joining analysis postulated the presence of two median vectors between I. bistriata haplotypes from adjacent localities, suggesting that genetic isolation between them is unrelated to geographic distance. These results confirm previous molecular differences suggesting that I.bistriata is a composite of several taxa. The karyotype of Isothrix negrensis also differs from those reported for I. bistriata and I. pagurus. In relation to the external morphology I. negrensis differs from I. bistriata by the overall darker dorsal coloration, head darker than dorsum, presence of a longitudinal line of blackish hairs in the proximal part of the tail extending until near to its base, dorsal surface of hind feet covered with yellowish hairs and stripe of light hairs in the head reduced to a small patch.