The synthesis and turnover of triglyceride in adipose tissue involves enzymes with preferences for specific fatty acid classes and/or regioselectivity with regard to the fatty acid position within the glycerol moiety. The focus of the present study was to characterize both the fatty acid composition and their positional distribution in triglycerides of biopsied human subcutaneous adipose tissue using 13C NMR spectroscopy. The triglyceride sn2 position was significantly more enriched with monounsaturated fatty acids compared to the sn1,3 sites, while saturated fatty acids abundance was significantly lower in the sn2 position compared to that of sn1,3. Furthermore, the analysis revealed significant positive correlations between the total fraction of palmitoleic acid with both BMI and HOMA-IR scores. Additionally, we established that 13C NMR chemical shifts for ω−3 signals, centered at 31.9 ppm, provided superior resolution of the most abundant FA species, including palmitoleate, compared to the ω−2 signals that were used previously. 13C NMR spectroscopy reveals for the first time a highly non-homogenous distribution of FA in the glycerol sites of human adipose tissue triglyceride and that these distributions are correlated with different phenotypes such as BMI and insulin resistance.