Mercury (Hg) is one of the most hazardous pollutants released by humans and is of global environmental concern. Mercury causes oxidative stress and strong cellular damages in plants, which can be attenuated by the biosynthesis of thiol-rich peptides (biothiols), including glutathione (GSH) and phytochelatins (PCs). We analysed Hg tolerance and speciation in five Arabidopsis thaliana genotypes, the wild-type Col-0, three knockdown gamma-glutamylcysteine synthetase (gamma ECS) mutants and a knockout PC synthase (PCS) mutant. Mercury-PC complexes were detected in roots by HPLC-ESI-TOFMS, with its abundance being limited in gamma ECS mutants. Analysis of Hg-biothiol complexes in the xylem sap revealed that HgPC2 occurs in wild-type Col-0 Arabidopsis, suggesting that Hg could be translocated associated with thiol-rich metabolites. Twenty genes involved in sulphur assimilation, GSH and PCs synthesis were differentially expressed in roots and shoots, implying a complex regulation, possibly involving post-translational mechanisms independent of GSH cellular levels. In summary, the present study describes the importance of biothiol metabolism and adequate GSH levels in Hg tolerance and identifies for the first time Hg-PC complexes in the xylem sap. This finding supports the notion that Hg-biothiol complexes could contribute to Hg mobilisation within plants. Highlights: • Low glutathione (GSH) in Arabidopsis γ-glutamylcysteine synthase (γECS) cad2-1, pad2-1, rax1-1 mutants resulted in poor Hg tolerance. • Those mutants had also less abundant Hg-phytochelatin (PC) complexes, as showed HPLC-ESI-MS(TOF) analysis. • For the first time, occurrence of HgPC2 complexes in xylem sap of Hg-treated Arabidopsis suggests long distance Hg-PCs transport. • There was differential sulphur-assimilatory genes expression, but complex regulation independent of GSH levels may occur.