Thlaspi caerulescens is increasingly acknowledged as the best model for the study of heavy metal hyperaccumulation in plants. As the first attempt to apply proteomics on this species, three T. caerulescens populations with pronounced variation in the uptake, root to shoot translocation and tolerance of Zn and Cd were compared. Total soluble proteins were separated using 2-dimensional electrophoresis, stained with SYPRO Orange, and intensity values and quality scores obtained for each spot using PDQuest software. The statistical methods applied were the analysis of variance (ANOVA) and Kruskal-Wallis non-parametric test to identify individual protein spots with significantly different expression levels, and principal component analysis (PCA) to identify groups of proteins which, in combination, had different expression levels among the experimental treatments. Based on ANOVA, the expression differed most between the populations, the effects of exposures being less pronounced. The PCA plots for root and leaf protein spots significantly affected only by population showed clear separation between the three populations. The PCA plots for protein spots where protein expression was significantly affected by both population and exposure also indicated separation between the populations. A number of proteins which showed statistically significant differences in expression were identified by mass spectrometry.