<p>To evaluate the outcomes of deep anterior lamellar keratoplasty (DALK) using stromal tissues obtained during Descemet membrane endothelial keratoplasty (DMEK) and to compare the effectiveness of fresh versus three-day–stored grafts. This two-center retrospective study included 42 eyes of 42 patients who underwent DALK using DMEK-derived stromal tissues. Patients were divided into two groups: Group 1 (<i>n</i> = 23), in which fresh stromal tissue was used on the day of surgery, and Group 2 (<i>n</i> = 19), in which stromal tissue stored under cold-chain conditions—defined as transportation with the tissue temperature consistently maintained at approximately 4&#xa0;°C—for three days was used. Preoperative and postoperative evaluations included best-corrected visual acuity (BCVA), intraocular pressure, slit-lamp and fundus examination, and corneal tomography (Pentacam HR; Oculus, Germany). Demographic data, intraoperative characteristics, postoperative graft transparency, and complications were compared between groups. The groups were similar regarding demographic features, surgical indications, anesthesia type, and suture technique (all <i>p</i> &gt; 0.05). Graft transparency did not differ significantly between the groups on postoperative day 1 or at the last visit (<i>p</i> = 0.597, <i>p</i> = 0.890). Double anterior chamber formation occurred in 3 eyes (15.8%) in Group 1 and 6 eyes (26.1%) in Group 2 (<i>p</i> = 0.418). Other complications, including glaucoma, cataract, epithelial defects, and keratitis, occurred at similar rates (all <i>p</i> &gt; 0.05). BCVA, keratometric, pachymetric, and densitometric parameters significantly improved postoperatively in both groups, with no intergroup difference (all <i>p</i> &gt; 0.05). Stromal tissue obtained during DMEK is a safe and effective graft source for DALK, whether used fresh or after short-term storage. This technique optimizes donor cornea utilization and may help alleviate the global donor shortage.</p>