This work aimed to study the changes in starch and isolated starch resulting from the conversion of corn to tortilla, focusing on the orthorhombic crystal structure and its association with resistant starch. Scanning electron microscopy images show whole, partially, and completely damaged starch granules in nixtamalized corn, masa, and tortillas. More importantly, whole isolated starch granules were found in nixtamal, masa, and tortillas. Transmission electron microscopy shows the presence of nanocrystals with orthorhombic structures in isolated starch. Some of them remained almost undamaged during the nixtamalization process. The X-ray patterns showed orthorhombic crystals in nixtamal, masa, and tortilla and their isolated starches. The RS increased from 2.61 to 5.31 % from corn to tortilla and from 2.52 to 5.61 % for isolated starches from corn and tortilla during the traditional nixtamalization process. The results suggest that the nanocrystals in corn to tortilla are part of RS2.