Epididymal sperm may be available as a source of spermatozoa for artificial insemination and in vitro embryo production in many species. The aim of the study was to investigate epididymal sperm freezing on spermatological parameters of Anatolian buffaloes and in vitro evaluation of frozen epididymal sperm for assisted reproductive biotechnologies. The testes and epididymides were collected from 10 Anatolian buffalo bulls (about 3-4 years of age) in Samsun Province. The testes and epididymides from each bull were removed immediately after slaughtering and transported to the laboratory on ice (+4 degrees C) within 40 min for further processing. After morphological measurements of testes and epididymides were determined, the spermatozoa were collected from cauda epididymides. The percentage of sperm progressive motility and abnormal spermatozoa of fresh and post-thaw sperm samples were evaluated. There was no significant difference between fresh and frozen-thawed sperm samples for progressive sperm motility and head-acrosome anomalies of spermatozoa. The middle part (P<0.001), total (P<0.001) and total (P<0.001) abnormal spermatozoa rates of frozen-thawed sperm samples were higher than those of fresh sperm samples. In conclusion, collected spermatozoa from cauda epididymides of Anatolian buffalo bull may be used as fresh or frozen for artificial insemination, in vitro embryo production or intra cytoplasmic sperm injection.