Genetic studies constitute a powerful approach to study the introduction and expansion of invasive species. Senecio inaequidens DC is one of the Europe's fastest plant invaders and is suspected to use railway corridors to expand. In France, the species has first been observed in the city of Paris in the late 1990's and today it is present throughout the region. In order to assess the role of railways in S. inaequidens invasion, we used innovative multivariate analyses to study population genetic diversity of this allotetraploid species. We analyzed the genetic diversity at 11 microsatellite markers of a total of 450 individuals sampled at 15 locations along railways connecting the city of Paris to the surrounding suburban/rural areas. Geographical distances among locations ranged from 1 to 83 km with four locations within Paris at the departure train stations of the national/international train lines studied. To our knowledge, studies on invasive allotetraploid plants where data were kept in the tetraploid format are still scarce. Our analyses revealed substantial genetic diversity and clear genetic differentiation among some populations, with a genetic gradient detected along one railway line. Results also showed no genetic bottleneck between Parisian populations and the historically introduced population located in South of France. Our study thus revealed the role of railways as a corridor among S. inaequidens populations within central Paris. However, connecting networks appeared more complex in the suburban and rural areas suggesting other potential vectors.