To report outcomes of femtosecond-assisted single-piece mushroom keratoplasty for the treatment of full-thickness corneal disease in pediatric patients with healthy endothelium. Femtosecond-assisted mushroom keratoplasty was performed in 8 eyes of 8 patients (age range, 8-17years) with central full-thickness corneal opacity. The single-piece mushroom-shaped graft consisted of a large anterior portion (9mm in diameter; 250μm in thickness) and a small posterior portion (6-6.5mm). Donor and recipient corneas were prepared using the WaveLight FS200 laser (Alcon Laboratories, Fort Worth, TX). The donor cornea was oversized by 0.2mm. Outcome measures were best spectacle-corrected visual acuity, spectacle refraction, topographic astigmatism, endothelial cell density, graft rejection, and graft failure at 1, 3, 6, and 12months. Mean best spectacle-corrected visual acuity at 1, 3, 6, and 12months was 0.28, 0.16, 0.13, and 0.10 logMAR; all patients achieved logMAR of at least 0.4 at 1, 3, 6, and 12months. The mean refractive cylinder was 2.6 D, and mean endothelial cell loss was 13.3% at 12months postoperatively. Two eyes had immunologic rejection episodes that were reversed with topical steroids. All corneas remained clear at final follow-up. Femtosecond-assisted mushroom keratoplasty is a viable surgical option for eyes of older pediatric patients with full-thickness corneal stromal disease and healthy endothelium. Mushroom keratoplasty combines the refractive advantage of a large keratoplasty with the immunologic advantage of a small keratoplasty. Single-piece femtosecond-assisted mushroom keratoplasty may have a mechanical advantage over regular penetrating keratoplasty.