Acuumulated evidence has indicated that roads are contributing to the spread of invasive exotic plants in numerous ecosystems by providing corridors for invasion and suitable habitat. However, little has been known about the effect of roads on the exotic plant soil seed banks (SSBs) which are important components of population dynamics, affecting the ecosystem resilience. Herein, we examined the effect of different roadside habitats and distance from roads on SSB of Eupatorium adenophorum (EA) towards revealing the regeneration and recruitment of its population. Importantly, our study will be remarkably helpful to realize the relationship between roads and exotic plant invasion and propose measurements for weed regulation. We performed the study at 23 sits along 9 roads of three different grades in southern and middle part of Yunnan where was invaded by EA. 10 cm×10 cm× 10 cm soil samples were taken along transects leading awy from the edges of roads at each sit. Totally, 55 transects were established and 374 soil samples were collected. Greenhouse germination method was applied to determine SSB storage and density. The results showed that there were abundant EA seeds stored in SSBs as well as the 9 roads with densities ranged from 3 152 to 25 225 ind/m². Averagely, the proportion of EA seeds reached 48.7% of the total SSBs. The grade of roads, landscape along roadsides and elevation showed significant effects on EA seed bank density: it increased with road grade; density of different roadside landscape together with the order of Open forest> Open shrubs>Abandoned land and slope>Forest; it reached its maximum within 1 700-1 900 m above sea level. The curve of both seed bank density and the proportion of the total number of transects seeds have only a single peak. Density of EA seeds at the nearest point of the roads was already very high, however, the highest density in any transects was usually found within 9 m distance to the roadside. Taken together, we can conclude that there exists a seed storage belt along with roadsides, and management of roadside habitats should be considered as a key to prevent and controll exotic plant invasion, and the maintenance of a dense canopy of native vegetation would benefit the regulation of EA.