Molecular dynamics simulations are combined with solid-state NMR measurements to gain insight into the macroscopic structural composition of the π-conjugated polymer poly(2,5-bis(3-tetradecyl-thiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT). The structural and dynamical properties, as established by the NMR analyses, were used to test the local structure of three constitutient mesophases with (i) crystalline backbones and side chains, (ii) lamellar backbones and disordered side chains, or (iii) amorphous backbones and side chains. The relative compositions of these mesophases were then determined from the deconvolution of the 1H and 13C solid-state NMR spectra and dynamic order parameters. Surprisingly, on the basis of molecular dynamics simulations, the powder composition consisted of only 28% of the completely crystalline mesophase, while 23% was lamellar with disordered side chains and 49% amorphous. The protocol presented in this work is a general approach and can be used for elucidating the relative compositions of mesophases in π-conjugated polymers.