Hybridization between invasive and native species accounts among the major and pernicious threats to biodiversity. The Mozambique tilapia Oreochromis mossambicus, a widely used freshwater aquaculture species, is especially imperiled by this phenomenon since it is recognized by the IUCN as an endangered taxon due to genetic admixture with O. niloticus an invasive congeneric species. The Lower Limpopo and the intermittent Changane River (Mozambique) drain large wetlands of potentially great importance for conservation of O. mossambicus, but their populations have remained unstudied until today. Therefore we aimed (1) to estimate the autochthonous diversity and population structure among genetically pure O. mossambicus populations to provide a baseline for the conservation genetics of this endangered species, (2) to quantify and describe genetic variation of the invasive populations and investigate the most likely factors influencing their spread, (3) to identify O. mossambicus populations unaffected by hybridization. Bayesian assignment tests based on 423 AFLP loci and the distribution of 36 species-specific mitochondrial haplotypes both indicate a low frequency of invasive and hybrid genotypes throughout the system, but nevertheless reveal evidence for limited expansion of two alien species (O. niloticus and O. andersonii) and their hybrids in the Lower Limpopo. O. mossambicus populations with no traces of hybridization are identified. They exhibit a significant genetic structure. This contrasts with previously published estimates and provides rather promising auspices for the conservation of O. mossambicus. Especially, parts of the Upper Changane drainage and surrounding wetlands are identified as refugial zones for O. mossambicus populations. They should therefore receive high conservation priority and could represent valuable candidates for the development of aquaculture strains based on local genetic resources.