The systematic status and geographic distribution of spiny mice of the genus Acomys I. Geoffroy, 1838 in Northern Tanzania is uncertain. This study assesses the systematic and geographic distribution of Acomys from Northern Tanzania using a multidisciplinary approach that includes molecular, cytogenetic, traditional and geometric morphometric analyses, and classical morphology of the same individuals. The molecular analysis was based on 1140 base pairs (bp) of the mitochondrial cytochrome b and 1297 bp of the nuclear interphotoreceptor retinoid binding protein (IRBP) gene sequences. These data were subjected to phylogenetic analyses using Maximum likelihood, Bayesian, Maximum parsimony, and Minimum evolution analyses. The cytogenetic analysis included G-banding of metaphase chromosomes. The morphometric analyses included univariate and multivariate analyses of traditional morphometric measurements of the cranium and mandible, and of geometric morphometric two-dimensional landmarks of the dorsal, ventral, and lateral views of the cranium, and lateral view of mandible that included thinplate spline (TPS) analysis. The classical morphology included examination of external, cranial and mandibular morphology. Results of all these multidisciplinary analyses were congruent and provide evidence for the occurrence of two sympatric species of Acomys in northern Tanzania, namely, the previously recorded A. wilsoni (2n = 62) and a newly recorded A. cf. percivali (2n = 58). These results that also represent the first reported mitochondrial cytochrome b and nuclear IRBP gene sequences and karyotype for A. cf. percivali, increases the number of species known to occur in Tanzania from four to five. However, the mitochondrial cytochrome b data that included GenBank sequences from the type locality in Kenya suggest that A. wilsoni may not be monophyletic. Ecologically, the two species seem to partition their niches with A. cf. percivali being found in well-covered habitats with thorn bushes, rocky and mountainous areas, and A. wilsoni being found in open semi-arid grasslands as well as in rice fields. The two species appear to be isolated by complex natural barriers formed by the Great East African Rift Valley whose geological features have generally been associated with active rodent speciation. However, the present results need further multidisciplinary investigation involving extensive sampling and examination of topotypical material.