Purpose The study aims to compare the impact of non-mechanical excimer laser-assisted (EXCIMER) and femtosecond laserassisted (FEMTO) trephinations on graft endothelial cell density (ECD) and graft thickness before and after suture removal following penetrating keratoplasty (PK). Methods The inclusion criteria for this prospective, randomized, clinical study were as follows: (1) surgeries performed by one surgeon; (2) primary central PK; (3) keratoconus (KC) or Fuchs' dystrophy (FUCHS); (4) no previous intraocular surgery; (5) graft oversize, 0.1 mm; and (6) 16-bite double-running suture. In 68 eyes of 68 patients (mean age: 53.3 ± 19.8 years), PK was performed using either 193-nm MEL70 excimer laser (BEXCIMER^: 17 KC, 18 FUCHS) or 60-KHz femtosecond laser (BFEMTO^: 17 KC, 16 FUCHS) trephination. Specular microscopy (EM 3000) and pachymetry (EM 3000; Pentacam HR; Casia SS-1000) were performed before removing the first suture (11.4 ± 1.9 months) and after removing the second suture (22.6 ± 3.8 months), but before any additional ophthalmic surgery. Results ECD did not differ significantly (P ≥ 0.436) between EXCIMER and FEMTO either with Ball-sutures-in^(1887 ± 409 vs. 1886 ± 438) or with Ball-sutures-out^(1703 ± 379 vs. 1737 ± 362). Central corneal thickness and corneal thickness at the thinnest point of the cornea did not differ significantly between EXCIMER and FEMTO either with all-sutures-in (P ≥ 0.096 and P ≥ 0.653) or with all-sutures-out (P ≥ 0.636 and P ≥ 0.717). Conclusions EXCIMER and FEMTO trephinations from the epithelial side seem to have no disadvantages regarding endothelial cell loss after PK, and both surgical procedures are safe for the endothelium. A larger sample size and longer follow-up are needed to evaluate the long-term impact of EXCIMER and FEMTO trephinations on ECD.