Abstract: A method based on the coupling of sizeexclusion HPLC with ICP-MS was used for speciation analysis of Cd，Cu and Zn binding nonprotein thiols in extracts of Indian mustard (Brassica Juncea). In the presence of Cd， Cu and Zn stress， phytochelatins (PCs) were induced and metal ions in extracts were associated with the induced PCs. Four different species of (PC)3Cd(Cu，Zn), (PC)2Cd(Cu，Zn), GSHCd(Cu，Zn), CysCd(Cu，Zn) were detected in stems and roots, and their distribution patterns within plants was studied. The results indicated that there was a competition on the combination of Cd, Cu, Zn with GSH and Cys. A nonoxidizing (nitrogen) sample preparation environment and -70℃ preservation condition were employed to avoid the oxidation of sulfhydryl groups. This research may be useful for understanding transportation of heavy metals in hyperaccumulator.