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Cell wall bound and intracellular cadmium content was determined after exposition to 70\u2003<jats:italic>\u00b5<\/jats:italic><jats:sc>m<\/jats:sc> CdCl<jats:sub>2<\/jats:sub>, showing the main portion of the metal outside the cell. Nevertheless, imported cadmium was sufficient to cause significant changes in thiolpeptide metabolism and its transcriptional regulation. Modern analytical approaches enable new insights into phytochelatin (PC) distribution. A new rapid and precise UPLC\u2010MS method allowed high\u2010throughput PC quantification in algal samples after 1, 4, 24 and 48\u2003h cadmium stress. Initially, canonic PCs were synthesized in <jats:italic>C.\u2003reinhardtii<\/jats:italic> during cadmium exposition, but afterwards CysPCs became the major thiolpeptides. Thus, after 48\u2003h the concentration of the PC\u2010isoforms CysPC<jats:sub>2\u20103<\/jats:sub> and CysGSH attained between 105 and 199\u2003nmol\u2003g<jats:sup>\u22121<\/jats:sup> fresh weight (FW), whereas the PC<jats:sub>2\u20103<\/jats:sub> concentrations were only 15\u2003nmol\u2003g<jats:sup>\u22121<\/jats:sup> FW. The relative quantification of <jats:italic>\u03b3<\/jats:italic>\u2010glutamyl transpeptidase (<jats:italic>\u03b3<\/jats:italic>\u2010GT) mRNA suggests the generation of CysPCs by glutamate cleavage from canonic PCs by <jats:italic>\u03b3<\/jats:italic>\u2010GT. Furthermore, a homology model of <jats:italic>C.\u2003reinhardtii<\/jats:italic> phytochelatin synthase was constructed to verify the use of crystal structures from <jats:italic>Anabaena<\/jats:italic> sp. phytochelatin synthase (PCS) for docking studies with canonical PCs and CysPCs. From the difference in energy scores, we hypothesize that CysPC may prevent the synthesis of canonical PCs by blocking the binding pocket. 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