The objective of this study was to elucidate effects of formulation and process variables on the physical forms of freeze-dried myo-inositol. Physical properties of myo-inositol in frozen solutions, freeze-dried solids, and cooled heat-melt solids were characterized by powder X-ray diffraction (PXRD), thermal analysis (differential scanning calorimetry [DSC] and thermogravimetric), and simultaneous PXRD-DSC analysis. Cooling of heat-melt myo-inositol produced two forms of metastable anhydrate crystals that change to stable form (melting point 225 °C-228 °C) with transition exotherms at around 123 °C and 181 °C, respectively. Freeze-drying of single-solute aqueous myo-inositol solutions after rapid cooling induced crystallization of myo-inositol as metastable anhydrate (transition at 80 °C-125 °C) during secondary drying segment. Contrarily, postfreeze heat treatment (i.e., annealing) induced crystallization of myo-inositol dihydrate. Removal of the crystallization water during the secondary drying produced the stable-form myo-inositol anhydrate crystal. Shelf-ramp slow cooling of myo-inositol solutions resulted in the stable and metastable anhydrous crystal solids depending on the solute concentrations and the solution volumes. Colyophilization with phosphate buffer retained myo-inositol in the amorphous state. Crystallization in different process segments varies crystal form of freeze-dried myo-inositol solids.