Background and Aims Roads as corridors of seed or fruit spatial dispersal have major impacts on the establishment and spread of invasive species, but their precise role in population genetic variation remains poorly understood. The South American weed Mikania micrantha has spread rapidly across southern China since its introduction to the Shenzhen area in 1984. This study investigated how its genetic diversity is distributed along highways, and whether highways have acted as corridors for the rapid expansion of M. micrantha.Methods Twenty-seven roadside populations were sampled along four highways in southern China, and 787 samples were examined using 12 microsatellite markers. Variation in genetic diversity among populations was quantified and patterns of genetic differentiation were analysed.Key Results A high level of genetic diversity was found at both the species and the population levels in this selfincompatible plant (expected heterozygosity ¼ 0Á497 and 0Á477, respectively; allelic richness ¼ 2Á580 and 2Á521, respectively). The Wright F-statistic value among populations (0Á044, P < 0Á01) and the analysis of molecular variance (91 % of genetic variation residing within populations, 9 % among populations within highways and 0 % among the four highways) showed a relatively low level of genetic differentiation among populations, while the principal coordinate and cluster analyses also indicated a lack of clear geographical genetic structure among populations. The calculated N m value of 5Á5 signifies strong gene flow.Conclusions The pattern of genetic variation is consistent with facilitated dispersal along highways. The genetic admixtures among the roadside populations imply the occurrence of multiple population introductions during colonization. The long-distance dispersal of seeds associated with vehicular transportation on highways may have played important roles in shaping the genetic variation. This finding highlights the importance of highways as corridors for the spread of M. micrantha in southern China.