The American mink (Mustela vison) is known as a successful non-native species in Europe, impacting native species' population sizes and habitats. This study investigates the genetic structure and diversity of American mink populations in France over two decades (1997–2016). The analysis involves feral and farmed mink sampled from various regions, using ten autosomal microsatellite loci for genotyping. The objective is to identify the putative existence of genetic lineages, especially between feral and farmed individuals, and to assess changes in genetic structure over time. Results reveal high genetic diversity and inbreeding within populations, with evidence of genetic structure influenced by both farm releases and feral colonization. The study highlights the reflection of the genetic structure in farm populations in the feral populations within the first period (1997–2007), and a decline of a lineage over time in the second period (2007–2016) with the emergence of a new genetic cluster, potentially influenced by factors such as selection, phenotypic changes, and interactions with pathogens. Overall, this research contributes to the understanding of the dynamics of American mink populations in France and their genetic variability, emphasizing the importance of ongoing monitoring and management efforts to mitigate the impact of this invasive species, especially on endangered or/and endemic species such as European mink (Mustela lutreola) and Iberian desman (Galemys pyrenaicus).