Two subspecies of waterbuck (Kobus ellipsiprymnus), common (Kobus ellipsiprymnus ellipsiprymnus) and defassa (Kobus ellipsiprymnus defassa), are recognized based on differences in rump pattern, coat colour and geographical distribution. These forms are parapatrically distributed with an area of range overlap in East Africa, where phenotypically intermediate populations occur. Variation in 478 bp of the mitochondrial DNA control region and 14 polymorphic microsatellite loci were used to describe the genetic structure and phylogeographical pattern of the species, and to assess if the intermediate populations are the results of hybridization. In total, 186 individuals from 11 localities were analysed. A high degree of genetic differentiation was found between subspecies, although this was most evident from the microsatellite data. Hybridization was suggested in the phenotypically and geographically intermediate Nairobi NP population in Kenya. A neighbour-joining (NJ) tree based on microsatellite population genetic distances grouped Nairobi between the common and defassa populations, and a Bayesian analysis clearly showed introgression. Individuals sampled in Samburu NP, Kenya, had a common waterbuck phenotype, but introgression was suggested by both markers. Although a high degree of maternal defassa input was indicated from the sequence data, the Samburu population grouped with the common waterbuck in the microsatellite population genetic distance tree, with high support. Analyses of linkage disequilibrium and maximum-likelihood estimates of genetic drift suggested that admixture between subspecies is a recent event. The fact that introgression is limited between subspecies could be caused by chromosomal differences, hindering gene flow between common and defassa waterbuck.