As a result of water pollution and river management practices, most Flemish bullhead populations are nowadays both small and isolated. Consequently, due to the loss of genetic variation associated with this, several populations are threatened with extinction. In order to evaluate the impact of human activities on these populations, and to develop appropriate conservation strategies, different approaches, considering different processes and endpoints, were combined. We determined conservation units using both highly variable microsatellite markers and mitochondrial DNA sequences. These data were also used to assess genetic variation and population differentiation. A series of challenge tests was used to investigate the populations' physiological condition. Furthermore, population characteristics such as population size, age distribution, condition factor and mobility were determined. The results of these different studies allowed us to evaluate the severity of the problem and to formulate conservation strategies for this species.