Plant glutathione S-transferases (GSTs) play important roles in stress tolerance and detoxification metabolism. We cloned two Phi GST genes (PtoGSTF1 and PtoGSTF2) from Populus tomentosa. PtoGSTF1 and PtoGSTF2 encode a protein of 215 and 218 amino acid residues, with a calculated molecular mass of 24.32 and 24.57 kDa, respectively. Genomic sequence analysis showed that PtoGSTF1 and PtoGSTF2 contained 2 introns. RT-PCR revealed PtoGSTF1 and PtoGSTF2 constitutively expressed in P. tomentosa. Recombinant PtoGSTF1 and PtoGSTF2 proteins were overexpressed in E. coli and purified by Ni-affinity chromatography. PtoGSTF1 and PtoGSTF2 showed enzymatic activities towards the substrates CDNB, NBD-Cl, NBC and Cum-OOH. The affinity to substrate NBD-Cl was 2.5-fold higher for PtoGSTF2 than PtoGSTF1, but the catalytic efficiency to NBD-Cl was 56.85-fold greater for PtoGSTF1 than PtoGSTF2. PtoGSTF1 showed greater thermal stability than PtoGSTF2. These results indicate the functional divergence between PtoGSTF1 and PtoGSTF2.