Although heavy‐metal biomonitoring in aquatic environments has been traditionally based on chemical analyses, these do not reflect the impact on organisms; because of this, international environmental agencies have proposed the use of biomarkers. In this study Cr, Ni, Cu, Zn and Pb were bio‐monitored in different species of aquatic plants using Total Reflection X‐Ray Fluorescence (TXRF) spectrometry. The relation between each metal with phytochelatins (PCs) was also assessed in order to establish the use of PCs as biomarkers for water pollution contaminants. Samples of Lemna gibba, Myriophyllum heterophyllum, Arenaria paludicola, Hydrocotyle ranunculoides and Eichhornia crassipes collected in natural aquatic environment were analyzed. The results showed significant differences in metal concentration between species both in shoots and roots: Cr, Ni, Cu and Pb were greater in roots in the majority of samples; the maximum content of Pb (7.92 mg/kg) was found in roots of E. crassipes, Cr (7.54 mg/kg), Ni (5.96 mg/kg) in roots and Zn (64.10 mg/kg) and Cu (60.55 mg/kg) in shoots of H. ranunculoides, which showed the heaviest metal concentration. Glutathione (GSH) and the PCs concentrations were also ascertained. Differences between species and parts of the plants were also found, although the tendency was different as regards elemental concentration. Marked differences were detected in metal absorption and sensitivity to produce PCs of the aquatic plants. Pb was the metal with the least concentration in the plants, but, it was the only element that showed a direct correlation (p < 0.05, r > 0.9) with the PCs induction.