Poly-l-alanine (PLA) sequences are key elements of the crystalline domains of spider dragline and wild silkworm silks. In the present work, 1H spin-diffusion two-dimensional (2D) correlation NMR spectra were observed for selectively deuterated (Ala)3 and (Ala)4 crystals to develop the analytical method for the structure of PLA sequences. The build-up curves of the cross peaks for three kinds of 1H pairs in selectively deuterated (Ala)3 and (Ala)4 crystals were observed to obtain spin-diffusion rate constant k j, k from relaxation master equations P i, j(τm). The k j, k values subsequently lead to effective interproton distance r j, keff (obs) values for individual proton-proton pairs, which include intra- and intermolecular contributions. The r j, keff (obs) values were compared to r j, keff (calc) values obtained from the experimentally determined atomic coordinates of antiparallel (AP) β-sheet (Ala)3 and (Ala)4 and parallel (P) β-sheet of (Ala)3 and (Ala)4 crystals. The agreement between the r j, keff (obs) and r j, keff (calc) values was good for AP β-sheet (Ala)3 and (Ala)4 crystals but poor for P β-sheet (Ala)3 and (Ala)4 crystals. These deviations were obtained from the interproton distances of the interchain contributions due to different packing arrangements. The packing arrangements of the PLA region are important when considering the relevant structure and the mechanical properties of silks.