Population units that merit separate management and are of conservation concern have been called evolutionary significant units. Two divergent lineages of the European rabbit Oryctolagus cuniculus occur naturally in Spain, with a well-marked geographical distribution. We analysed the frequency and importance of rabbit translocations in central-southern Spain and whether this practice, carried out by hunters and conservationists, could cause the mixture of two clearly different evolutionary significant units. We carried out interviews in 1993 and 2002 at 60 locations to determine the presence and intensity of translocations during both decades. The distribution of the lineages was obtained using mtDNA analysis of hunted rabbits in 2003-2005. We demonstrate that rabbit translocation was used frequently in the 1980s and increased in the 1990s. Up to 43% of the studied areas translocated rabbits in the latter decade, whereas only 25% did so in the 1980s. Our results show that neither the origin of the introduced rabbits nor their genetic lineage were taken into account in most of the translocations. We found rabbits of lineage A in several localities within the distribution area of lineage B, and vice versa, probably as a consequence of translocations. The distribution of both lineages is likely to have been altered by human activity and this could represent the loss of the results of 2 million years of genetic differentiation with possible attendent ecological consequences. Consequently, authorities should more closely regulate rabbit translocations and convey to both hunters and conservationists the importance of not mixing the lineages by translocations.