Aim To optimise interfaces of endothelial buttons created with femtosecond (FS) lasers. Setting Department of Ophthalmology, Hôtel-Dieu Hospital, Paris, France. Methods Forty-two corneas were divided into five groups of various cutting patterns and a control group of 100 μm laser in situ keratomileusis flap creation. A single path full lamellar cut (500 μm) was applied to groups 1 and 2. The same full lamellar cut was applied twice to groups 3 and 4. Two successive lamellar cuts were performed in group 5 (350 and 150 μm). 60 kHz and 150 kHz were used respectively in groups 1, 3, 5, 6 and 2, 4. In each group, different laser settings were tested to obtain the best interface quality while delivering minimal energy to the stroma. The quality of stromal interfaces from created endothelial lenticules was observed using a scanning electron microscope. Results Stromal adherences persisted after both the single- and double-path procedure, creating central irregularities on the endothelial lenticule. Among all groups and settings tested, the double-layer pattern (group 5) with FS full lamellar cut parameters set for diameter (mm), depth (μm), energy (μJ) and spot size/step (μm) respectively on 9.0 mm, 350 μm, 2.1 μJ, 4:4 μm and 8.3 mm, 150 μm, 0.9 μJ, 4:4 μm created the smoothest interfaces with the best reproducibility. Conclusions Buttons for endothelial keratoplasty can be created with FS laser with a stromal interface quality comparable with that of refractive surgery.