Purpose To evaluate femtosecond laser-assisted corneal full-thickness trephination with orientation teeth and notches for penetrating keratoplasty (PKP). Design Interventional case series. Methods Four eyes of four patients aged 34 to 55 years underwent PKP using a femtosecond laser system enabling corneal trephination with orientation teeth and notches. Patients were affected by advanced keratoconus (n = 2) or postinfectious corneal scar (n = 2). Scanning electron microscopy (SEM) was performed to evaluate the cut quality of donor and recipient cornea. Results All procedures were completed without any complications. In all cases, the donor and the recipient cornea were cut with the same size of 8.0-mm diameter. A complete perforating cut was achieved in all eyes, and a correct intraoperative matching of teeth and notches was obtained. SEM of donor and recipient specimens showed a full-thickness cut with smooth and regular shape of orientation teeth and notches. Conclusions Orientation teeth and notches shaped PKP using the femtosecond laser technology is feasible and may offer further promising approaches toward customized trephination techniques.