Cold tolerance of alien invasive plants is a crucial determinant for their establishment and expansion into new cold environments. A close relationship between cold-tolerance level of 34 populations represented by 147 accessions and latitude, extreme lowest temperature, coldest month average temperature and invasion age revealed that cold-tolerance divergence is a key factor driving the spreading of Ageratina adenophora, a highly invasive plant in China, to subtropical areas northeastward from the first-colonized southwestern region. Four epialles of cold response regulator ICE1 were found ranging from 66 to 50 methylated cytosines, representing a 4.4% to 3.3% methylation rate and significantly corresponded to the lowest to highest cold-tolerance levels among those different populations. A comparative study of four geographically-distinct populations firstly demonstrated that ICE1 demethylation upregulated transcription level of CBF transcription pathway is responsible for this evolution. Those facts, combined with the variation of colt-tolerance and methylation found among three native and two other introduced populations, indicate that demethylation of a gene upregulating cold tolerance may be the underlying evolutionary mechanism allowing crofton weed to expand northwards in China.