Starch was isolated from banana starch and the retrogradation phenomenon was studied using diverse techniques, including an enzymatic measurement. Wide-angle X-ray scattering (WAXS) showed that the sample stored for 7 h presented small peaks and when the storage time increased the peaks increased in intensity. The type of diffraction pattern found in banana extrudates is typical of the A-type crystal polymorph. The crystallinity index from the diffractograms, showed a plateau after approximately 20 h of storage. The short-range order measurement with Fourier transform infrared (FTIR) spectroscopy showed that banana starch retrogradation reached a maximum value at approximately 11 h of storage, a value that agrees with the results obtained with differential scanning calorimetry (DSC), because the maximum enthalpy value (approximately 5 J/g) was calculated in the stored sample for 8 h, without changes in the stored samples for more time. Retrograded resistant starch values did not change after 12 h of storage, obtaining the maximum starch retrogradation level. FTIR, DSC, and the enzymatic technique showed the changes at the molecular level in starch during storage; in the case of WAXS, they determine the long-range order that explains the differences found in the starch retrogradation pattern measurement in banana starch.