Farnesyl diphosphate synthase (FPPS) is a key enzyme responsible for the supply of isoprenoid precursors for several essential metabolites, including sterols, dolichols and ubiquinone. In Saccharomyces cerevisiae, FPPS catalyzes the sequential condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), producing geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Critical amino acid residues that determine product chain length were determined by a comparative study of strict GPP synthases versus strict FPPS. In silico ΔΔG, i.e. differential binding energy between a protein and two different ligands-of yeast FPPS mutants was evaluated, and F96, A99 and E165 residues were identified as key determinants for product selectivity. A99X variants were evaluated in vivo, S. cerevisiae strains carrying A99R and A99H variants showed significant differences on GPP concentrations and specific growth rates. The FPPS A99T variant produced unquantifiable amounts of FPP and no effect on GPP production was observed. Strains carrying A99Q, A99Y and A99K FPPS accumulated high amounts of DMAPP-IPP, with a decrease in GPP and FPP. Our results demonstrated the relevance of the first residue before FARM (First Aspartate Rich Motif) over substrate consumption and product specificity of S. cerevisiae FPPS in vivo. The presence of A99H significantly modified product selectivity and appeared to be relevant for GPP synthesis.