Squalene synthase (SQS, EC 18.104.22.168) is a major enzyme in biosynthesis of isoprenoid (farnesyl pyrophosphate (FPP) squalene). In the present study, we have analyzed SQS enzymes of black cottonwood (Populus trichocarpa, hereafter Pt) and Masson's pine (Pinus massoniana, hereafter Pm) using bioinformatics tools. PtSQS and PmSQS sequences were found to have very similar physicochemical properties with "squalene/phytoene synthase" domain structure (PF00494). PtSQS sequence was 47.3 kDa weight and 413 amino acids long with a pI value of 6.86, while PmSQS was 46.6 kDa weight and 409 amino acids long with a pI of 7.92. Alignment of SQS protein sequences in 15 plant species showed a highly similar conserved pattern and included (DTVED81)-D-77 and (DYLED217)-D-213 motifs, which are rich in aspartic acids, for FPP binding sites. In phylogenetic tree, monocots and polycot were clearly separated from dicots with high bootstrap value (99 %). A total of 10 interaction partners were predicted for PtSQS and PmSQS proteins. Nine of them were hypothetical proteins (related with phytosterol biosynthesis), while one was putative uncharacterized protein. Similar 3D structures and identical binding sites were predicted for pine and poplar. In docking, FPP-PtSQS was found to make 8 H bonds with Asp81, Asp217, Glu80, and Gln206 residues in poplar with highest affinity while FPP-PmSQS made 7 H bonds with Arg49, Arg74, Ser48, and Val47 residues in pine with highest affinity. The results of this study will provide valuable theoretical knowledge for future studies of identification and characterization of SQS genes and proteins in various tree species and will provide an insight for studies of biotechnological manipulation of sterol biosynthesis pathway to enhance the plant stress tolerance and productivity.