Abstract: In the present study,bisulfite sequencing (BS-seq) was performed to detect alterations in DNA methylation of acid invertase gene promoters in Cu-tolerant (MP) and non-tolerant populations (NMP) of metallophyte Elsholtzia haichowensis under Cu treatment.We found that the vacuolar invertase gene (vINV) promoter of E.haichowensis was generally methylated in the CG site,whereas the cell wall invertase gene (cwINV) promoter displayed hypomethylation at the CG site.Methylation changes at a special CHG site of the vINV promoter and six CHH sites of the cwINV promoter induced by Cu stress were observed between the two populations.In addition,CG methylation was relatively conserved in the vINV and cwINV promoters under Cu stress,whereas methylation at the CHG and CHH sites was more prone to perturbation.Under Cu stress,methylation of these individual sites remained stable in the MP,but declined or increased drastically in the NMP.This implied that MP might have evolved modulating mechanisms due to long-term Cu stress,including changes in methylation patterns in specific DNA sequences and cytosine sites.