Bullous keratopathy is categorized as a corneal endothelial disease. However, pathological changes, including subepithelial fibrosis and the accumulation of extracellular matrix, have been detected in the corneal stroma of individuals with this condition. In vivo confocal microscopy allows the visualization of human corneal cellular structures and has provided information regarding how eyes are affected by various diseases. However, the determination of disease pathogenesis on the basis of in vivo confocal microscopic observations is problematic. We evaluated the structural alterations in the corneal stroma of eyes affected by bullous keratopathy using second harmonic generation microscopy and laser confocal immunofluorescence microscopy of whole-mount preparations. Using these approaches, we detected the transdifferentiation of keratocytes into fibroblasts and myofibroblasts at the anterior and posterior stroma and the presence of subepithelial fibrosis at the anterior stroma and disorganized collagen lamellae at the posterior stroma of the bullous keratopathy cornea. These changes were only detected in specimens from eyes with stromal edema lasting at least 12 months. Similar time-dependent changes were apparent by using in vivo confocal microscopy in the corneal stroma of patients with bullous keratopathy after performing a Descemet stripping automated endothelial keratoplasty surgery and were associated with an unfavorable outcome with regard to postoperative visual acuity. Our observations suggest that pathological changes in the corneal stroma of patients with bullous keratopathy are progressive and affect postoperative visual acuity after a Descemet stripping automated endothelial keratoplasty surgery is performed.