Purpose To compare the histological characteristics of keratinized versus non-keratinized onlay island flaps in an experimental rabbit model. Materials and Methods Sixteen male rabbits were randomly allocated into two experimental groups: keratinized and non-keratinized onlay island flaps. A defect was created in the ventral aspect of the penile urethra. In the keratinized group, a longitudinal island flap was harvested from the external prepuce and rotated to cover the urethral defect. In the non-keratinized group a transverse island flap was harvested from the inner prepuce. The animals were sacrificed after 2, 4, 8 and 12 weeks. Results The flaps were viable in all animals, and no deaths were associated with the procedure. Two urethrocutaneous fistulas were identified, one in each experimental group. A similar pattern of fibrosis was identified in both groups. The keratinized epithelium of the external prepuce kept its histological aspect and keratin production. Both keratinized and non-keratinized groups presented a slight decrease on the epithelial thickness, however without a statistically significant difference between groups. Conclusions In this short-term rabbit model, we observed that the stratified squamous keratinized epithelium from the external prepuce kept its keratin production. There was no statistical influence of the flap type on the mean epithelial thickness.