Sportive lemurs constitute a highly diverse endemic lemur family (24 species) for which many biogeographic boundaries are not yet clarified. Based on recent phylogeographic models, this study aims to determine the importance of two large rivers (the Antainambalana and Rantanabe) in northeastern Madagascar as species barriers for Lepilemur seali. The Antainambalana River was previously assumed to act as the southern border of its distribution. A total of 1,038 bp of the mtDNA of four individuals stemming from two adjacent inter-river systems south of the Antainambalana River was sequenced and compared to sequences of 22 described Lepilemur species. The phylogenetic reconstruction did not find support for either of the two rivers as species barrier for Lepilemur, as all captured individuals clustered closely with and therefore belonged to L. seali. However, a previously published sequence of an individual from a site south of our study sites belongs to a separate species. The southern boundary of L. seali must therefore be one of two large rivers further south of our study sites. The results suggest that L. seali may possess a relatively large altitudinal range that enabled this species to migrate around the headwaters of the Antainambalana and Rantanabe Rivers. Previous phylogeographic models need to be refined in order to incorporate these findings, and more species-specific altitudinal range data are urgently needed in order to fully understand the biogeographic patterns of lemurs on Madagascar.