The de novo synthesis of triglyceride (TG) fatty acids (FA) and glycerol can be measured with stable isotope tracers. However, these methods typically do not inform the contribution of a given substrate to specific pathways on these synthetic processes. We integrated deuterated water (HO) measurement of de novo lipogenesis (DNL) and glycerol-3-phosphate (GLY) synthesis from all substrates with a C nuclear magnetic resonance (NMR) method that quantifies TG FA and glycerol enrichment from a specific [U-C]precursor. This allowed the [U-C]precursor contribution to DNL and GLY to be estimated. We applied this method in mice to determine the contributions of fructose and glucose supplemented in the drinking water to DNL and GLY in liver, mesenteric adipose tissue (MAT) and subcutaneous adipose tissue (SCAT). In liver, fructose contributed significantly more to DNL of saturated fatty acids (SFA) and oleate as well as to GLY compared to glucose. Moreover, its contribution to SFA synthesis was significantly higher compared to that of oleate. MAT and SCAT had lower fractional rates of total DNL and GLY compared to liver and glucose was utilized more predominantly than fructose for TG synthesis in these tissues. This novel HO/C integrated method revealed for the first time, tissue specific selection of substrates for DNL, particularly fructose in regard to glucose in liver. Also, this approach was able to resolve the distribution of specific FAs into the TG sn2 and sn1,3 sites. This stable isotope integrated approach yielded information so far uncovered by other lipidomic tools and should powerfully assist in other nutritional, pathological or environmental contexts.