A survey of cotton (Gossypium hirsutum) volunteers was conducted along the 1400 km of a cottonseed transport route in north-east Australia to determine the extent of naturalisation from spilt seed. In each year, 1% of the road distance was inspected for volunteer plants. The survey results were juxtaposed with the eco-climatic suitability predictions from inferential modelling. Over 3 years, 22 cotton plants were found at eight sites. Within the cotton production region, volunteers averaged 2.28 plants km-1 of road; their most likely source was seed cotton spilt during the previous harvest. Further north, three plants were found over 3 years, at an average density of 0.089 plants km-1 of road; all three plants were found in locations with a positive Ecoclimatic Index. No secondary spread was detected. Roadside slashing reduced plant survival and the potential to produce seed. In the wet tropics, weed competition and slashing prevented volunteers establishing. The surveys indicate that roadsides in north-east Australia are a hostile environment for the establishment of cotton plants, with no evidence of naturalisation or secondary spread. Current transport practices utilizing fully covered loads present negligible risk of producing naturalised populations of cotton (either transgenic or non-transgenic) on roadsides in north-east Australia.