Plant stigmasterol synthesis involves the partaking of many genes. In the present study, we performed computational comparison of the genes smt2, smo2, ste1, dwf5, dwf1 and c-22 sterol desaturase involved in the phytosterol biosynthetic pathway in different plants. Eleven plants belonging to different botanical families were selected and the gene sequence was retrieved from the KEGG pathway database. Nucleotide sequence homology was observed by Clustal W analysis and phylogeny was studied using MEGA 5 software. Clustal W and MEGA 5 analysis was done for amino acid sequence also. The maximum conserved regions were used in Prosite scan analysis. To determine the domains in protein sequence of the aforesaid genes in eleven plants, Pfam domain database search was performed. Phyre 2 software was used to develop 3D protein models. All the monocots used in the study belong to Poaceae family and hence in our phylogenetic analysis of phytosterol biosynthetic genes showed 90% sequence homology among the plants with same point of origin. The dicot plants chosen for the study belong to different families and hence the genes showed a homology percentile of less than 80.Pfam studies revealed SMT2 protein with a Methyl transf 11 and Sterol MTC domains. SMO2 and STE-1 proteins showed a common FA hydroxylase domain. DWF-5 was found to have domain structure of ERG4/ERG24 family, whereas, DWF-1 showed presence of FAD binding 4 domain. P450 structural domain was represented by sterol c-22 desaturase proteins. The 3D structure prediction revealed structural similarity among all eleven plants for protein involved in the plant stigmasterol biosynthesis.