The present study sets out to assess patterns of evolutionary diversification in central African duikers (subfamily Cephalophinae). The sampling strategy consisted of collecting geo-referenced duiker feces across 43 sites and seven countries. However, several challenges related to the use of non-invasive samples needed to be addressed prior to large scale DNA amplification. First, the best storage method for obtaining DNA from fecal samples needed to be established. Our study revealed that while silica is best for nuclear microsatellite analyses, RNAlater is the best storage medium for maximal mitochondrial amplification. Moreover, extracting DNA as early as possible always provided the best results. Second, since it is impossible to determine the species identity of duiker feces solely based on their morphology, a simple and reliable molecular method was needed. A tree-based approach based on ~650 base pairs of the control region amplified from reference samples was found to be the most reliable method to recover the identity of unknown samples. Third, for fine scale analyses of population genetic structure, a set of twelve nuclear microsatellites were assembled from existing bovid data. These microsatellites markers were chosen because they are very polymorphic, cross amplify among targeted taxa, co-amplify with combined markers of the same multiplex, and are powerful enough for individual identification. Patterns of mitochondrial and nuclear microsatellite variation were used to test two important hypotheses of diversification in the tropics: the Pleistocene refugia and the riverine barrier hypotheses. Analyses of historical and contemporary population genetic structure were carried out on the three most abundant species in our sampling area: the bay duiker (C. dorsalis), the Peter’s duiker (C. callipygus), and the blue duiker (P. monticola) using mitochondrial and nuclear markers described above. These data show that (1) southwest Nigeria and southwest Cameroon comprise genetically distinct populations in C. callipygus and P. monticola species, (2) signatures of demographic expansion for all three taxa are broadly coincident with the location of hypothesized upland refugia in Gabon and Equatorial Guinea and (3) the Sanaga, Ogooué, and Sangha rivers may constitute a partial riverine barrier and/or act as fluvial refugia for duikers.