The purpose of this study was to assess the usefulness of transperineal ultrasonography in identifying the internal fistula in cases of imperforate anus. Transperineal ultrasonography was performed in 19 infants (13 neonates and 6 older infants; 13 were male and 6 were female) with imperforate anus to identify the internal fistula. Sagittal plane images were obtained through the anal dimple, and the internal connection of the rectal fistula was traced. The ultrasonographically traced internal fistula was compared with that observed on distal loopography after colostomy or with surgical findings. The internal fistula was identified as a hypoechoic linear tract, containing linear echogenicity in some cases. Of 19 patients, internal fistulas were correctly identified in 16 patients; these were rectourethral (n = 12), rectovaginal (n = 1), rectovestibular (n = 1), rectovesical (n = 1), and rectocloacal (n = 1). In three patients, internal fistulas were incorrectly defined; these cases consisted of rectovestibular (n = 2) and rectovaginal (n = 1) fistulas. Internal fistulas were correctly identified in all of the 13 male patients and in 3 of 6 female patients. Transperineal ultrasonography is an excellent diagnostic modality to define the type of the internal fistula in imperforate anus.