The objective of this study was to develop a color flow doppler ultrasound-guided technique for the pudendal nerve block in male donkeys. A detailed anatomical dissection was performed in five donkey cadavers to obtain the precise details of pudendal nerve and its relations and ramifications, detect the pudendal nerve blood vessels and to determine the optimal point for local anesthetic injection. The pudendal nerve in six male native breed donkeys was blocked successfully with local anesthetic solution. The injection site was located with the aid of ultrasound. Ten milliliters of local analgesic solution were injected via 20-cm, 18-gauge needle. Anesthetic effect was evaluated after bilateral pudendal nerve blocks. A feasible, applicable and highly accurate technique to locate the pudendal nerve was developed. Satisfactory anesthesia of the anus, perineum, penis, prepuce and glans penis was achieved while the animal in a steady standing position