Purpose. To compare different methods of glycerol-preserved corneas intended for deep anterior lamellar keratoplasty (DALK). Methods. We analyzed transparency, transmittance, thickness, biomechanics, morphology, and antigenicity of donor corneas preserved by four different glycerol-based methods (n = 6 per group) for 3 months, as follows: tissues in anhydrous glycerol without aluminosilicate molecular sieves at room temperature (GRT); tissues in anhydrous glycerol with aluminosilicate molecular sieves at room temperature (SRT); tissues in anhydrous glycerol without aluminosilicate molecular sieves at -78°C (G78); and tissues in anhydrous glycerol without aluminosilicate molecular sieves at -20°C (G20). Results. Slit lamp images and transmittance curves obtained by spectrophotometer show that the G78 cornea was the most transparent tissue. Stress-strain behavior indicated that corneas in the G78 group were the most pliable, and SRT corneas were the stiffest. Electron microscopy analysis indicated that corneal cytoarchitecture and keratocyte integrity was destroyed in all glycerol-preserved corneas. Disorganized stromal collagen fibers were evident in groups stored at RT. Especially in SRT corneas, parallelism was lost, fibrils were extremely tortuous and discontinuous, and widespread fibril degeneration could be found. Antigenicity of tissue, assessed via immunohistochemistry for CD45-positive cells, HLA-ABC and HLA-DR, was lowered after glycerol preservation relative to fresh cornea tissues, and immunoreactivity was located mainly on corneal epithelium and limbus rather than stroma. Conclusions. Anhydrous glycerol preservation without molecular sieves in a -78°C freezer was the best method to obtain DALK-eligible tissues that were both transparent and pliable. © 2012 The Association for Research in Vision and Ophthalmology, Inc.