Glutathione transferases (GSTs) represent a superfamily of multifunctional proteins which play crucial roles in detoxification processes and secondary metabolism. Instead of promoting the conjugation of glutathione to acceptor molecules as most GSTs, members of the Lambda class (GSTLs) catalyze deglutathionylation reactions via a catalytic cysteine residue. Three GSTL genes (Pt-GSTL1, Pt-GSTL2, Pt-GSTL3) are present in Populus trichocarpa, but two transcripts, differing in their 5’ extremities, were identified for Pt-GSTL3. Transcripts for these genes were primarily found in flowers, fruits, petioles and buds, but not in leaves and roots, suggesting roles associated with secondary metabolism in these organs. The expression of GFP fusion proteins in tobacco showed that Pt-GSTL1 is localized in plastids, whereas Pt-GSTL2 and Pt-GSTL3A and B are found in both the cytoplasm and the nucleus. The resolution of Pt-GSTL1 and Pt-GSTL3 structures by X-ray crystallography indicated that although these proteins adopt a canonical GST fold quite similar to that found in dimeric Omega GSTs, their non-plant counterparts, they are strictly monomeric. This might explain some differences in the enzymatic properties of both enzyme types. Finally, from competition experiments between aromatic substrates and a fluorescent probe, we determined that the recognition of glutathionylated substrates is favoured vs non-glutathionylated forms.