Azolla is a floating aquatic fern, having amazing capacity for concentrating toxic heavy metals. Metallothioneins (MTs) and phytochelatins (PCs) are well-defined heavy metal-binding ligands in plants. Bioaccumulation potential of different Azolla species varies according to their heavy metal ions. Therefore, the accumulation of Ni, Zn, Cu, and Cd was studied in A. pinnata , A. filiculoides, and a sample taken from Anzali wetland. Moreover, the expression of metallothionein and phytochelatin synthase encoding genes was examined at different metal concentrations. The highest level of Cu and Cd absorption was detected in A. pinnata, while the maximum amount of Ni and Zn absorption was observed in A. filiculoides and the sample taken from Anzali, respectively. The MT2 and PCS1 gene expression patterns were significantly induced by the heavy metal treatments, confirming their roles in phytoremediation potential of Azolla. However, as the results concerning heavy metal accumulation and gene expression vary in different species, only specific species of Azolla can be used for special purposes. It can be concluded that the Azolla is a good candidate for phytoremediation purposes, and the formation of phytochelatin-heavy metal complexes and their sequestration in vacuole are the main processes influencing susceptibility of Azolla to heavy metals. • Heavy metal accumulation of Azolla species and expression of MT2 and PCS1 genes were investigated. • The absorption ability of metals varied in different species. • MT2 and PCS1 gene expression were significantly induced by heavy metal treatments. • Manipulation of PCS1 and MT2 expression is one potential mechanism for phytoremediation. [ABSTRACT FROM AUTHOR]