Context: Biological invasions have caused dramatic changes in native biodiversity and ecosystem function. Studies of genetic variation and evolutionary changes are useful for understanding population dynamics during biological invasions, and shed light on management, prevention and restoration strategies. Aims: This study aimed to investigate the structure and genetic variability of American mink (Neovison vison), an invasive species in southern South America, introduced for fur farming in the 1930s. Methods: Samples from 153 mink were obtained from 12 locations in southern Chile to sequence the mitochondrial DNA (mtDNA) control region and to genotype 11 polymorphic microsatellite loci. Key results: The highest mtDNA diversity was detected in Puerto Cisnes, suggesting multiple introductions and/or the most probable area where mink was first introduced. The latter is also supported by microsatellite data, because a high percentage of individuals from different locations were assigned to this location. All other locations showed low or no mtDNA diversity, possibly due to founder effect. The results also indicate marked population structure, with three genetic clusters coincident with the main historical introduction points, with low dispersal among them. Conclusions: The results suggest that control strategies for American mink in southern Chile should be concentrated on these three genetically differentiated management units, and particularly on source populations and locations with low effective population size and restricted connectivity. Implications: Genetic approaches have been used for the management of numerous alien species worldwide. Recommendations delivered here for American mink control could also be implemented in other regions and for other invasive species with similar genetic diversity distribution and connectivity.