Abstract Objective To assess the use of tunica vaginalis graft plus onlay preputial island flap in urethral reconstructive surgery in rabbits through histopathology. Material and methods We developed an experimental model of urethroplasty that resembles one-stage complex hypospadias surgery with divided urethral plate. The tunica vaginalis graft is dorsally placed to recreate the urethral plate and the internal preputial island flap is placed onlay to complete the urethroplasty. Sixteen animals were divided into four equal groups and sacrificed at 2, 4, 8 and 12 weeks after surgery, the penis being sent for histopathological evaluation. Results There were no deaths related to the procedure or wound breakdown; all rabbits voided spontaneously after surgery. Two urethrocutaneous fistulae were found. Microscopically, good tissue integration was observed, the tunica vaginalis mesothelium was gradually replaced by a more stratified epithelial lining, similar to the urothelial lining of the native urethra. The stratified squamous non-keratinized lining of the internal preputial island flap also changed into a thinner epithelial lining with only 4–5 cell layers. Two urethral diverticula were found. Conclusion A tunica vaginalis graft placed dorsally plus an onlay internal preputial island flap was shown to be a successful technique for urethroplasty in an animal model.