In this work, we report and discuss on the use and limitations of the natural abundance deuterium two-dimensional NMR spectroscopy in polypeptide chiral and achiral aligning media in the studies of homogenous triglycerides at 14.1 T. As illustrative examples, two triglycerides with short and long alkyl chains were investigated: the 1,3-di(butanoyloxy)propan-2-yl butanoate or tributyrin (TB) and the 1,3-di(tetradecanoyloxy)propan-2-yl tetradecanoate or trimyristin (TM). If both flexible compounds are theoretically of Cs symmetry on average, according to the Altmann's definition (Proc. Roy. Soc., 1967, A298, 184.), the analysis of spectral data in terms of enantiotopic and diastereotopic discriminations shows noticeable differences related to their orientational ordering behavior inside the mesophases. Although from NMR analysis viewpoint, TB behaves as a Cs symmetry molecule as expected, the NMR results obtained for TM suggest a behavior that could be formally predicted for a C3v symmetry molecule on average. This conclusion was nicely supported by the comparison with the tri-n-propylorthoformate, a real C3v symmetry solute on average on the NMR timescale. This difference of effective orientational behavior could originate from the difference of size and shape between lateral and central alkyl chains of the solute molecule. Copyright © 2012 John Wiley & Sons, Ltd.