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Purpose: During donor tissue preparation for Descemet stripping automated endothelial keratoplasty (DSAEK), either microkeratome or femtosecond laser can be used for intrastromal cutting. We compared morphological and functional outcomes after DSAEK using both cutting techniques., Methods: In this retrospective study, 22 uneventful DSAEK surgeries were reviewed. Eight donor corneas were prepared for DSAEK using the VisuMax femtosecond laser (Carl Zeiss Meditec AG, Jena, Germany). Fourteen corneas were processed using an Amadeus II microkeratome (Ziemer Ophthalmic Systems AG, Port, Switzerland). The postoperative best spectacle-corrected visual acuity was measured. Furthermore, corneal optical coherence tomography scans (RTVue; Optovue, Fremont, CA) were conducted and analyzed for graft cornea thickness and posterior surface irregularities using regression analysis (SPSS; IBM, Chicago, IL) on a second-order polynomial curve as a model for the posterior surface., Results: The graft thickness was 166.3 ± 58.2 μm (mean ± SD) in the femtosecond laser group and 172.7 ± 48.2 μm in the microkeratome group. The best-corrected visual acuity of 0.48 ± 0.20 (logarithm of the minimum angle of resolution) in the femtosecond laser group was significantly poorer when compared with 0.33 ± 0.11 in the microkeratome group (P = 0.038). Moreover, the root mean square error between the posterior corneal surface and an ideal parabola surface was significantly higher in the femtosecond laser group (9.9 ± 2.2 μm) than in the microkeratome group (5.7 ± 2.2 μm; P < 0.001)., Conclusions: Our study underlines the current superiority of a microkeratome-assisted preparation of the stromal-endothelial lamella before DSAEK surgery compared with the curved interface femtosecond laser-assisted processing.