Background Micro-computerized tomography (micro-CT) is considered as an innovative non-invasive and high-resolution imaging technology. The current research aims to reconstruct the distribution of preputial vessels in different severity of rat congenital hypospadias model by micro-CT, and to provide an anatomic basis for the selection of preputial vessel pedicle flaps in surgery. Methods Pregnant rats were exposed to finasteride from gestational day 12 to 17. Depending on the position of the urethral meatus, the pups were divided into normal, mild hypospadias and severe hypospadias groups. Six months after birth, the preputial blood vessels were observed in vascular perfusion with Microfil (a silicone-based polymer) and scanned by micro-CT. CTvox and NRecon were utilized to reconstruct 3-dimentional (3D) images. A pathological analysis of the specimen was taken in order to determine the position of Microfil. Results The normal group and the mild hypospadias group had similar preputial image characteristics. At the junction of the inner and outer prepuce, the deep layer vessels of the superficial fascia were transversely distributed and formed a vascular ring-like structure. Among the severe hypospadias group, five had sufficient blood circulation while six had insufficient blood circulation. In sufficient blood circulation type, the ring-like vessels were found at the junction of the inner and outer prepuce similar to that of the normal and mild hypospadias group. However, only a small amount of capillary supply to this area in the insufficient type. Conclusion The junction of the inner and outer prepuce with abundant blood circulation was suitable to be a vascular pedicle flap. The tubularized preputial island flaps were consistent with the ring-like vessels area, and the original blood supply was retained to the greatest extent.