<p>If a male dies suddenly or requires castration, it may still be possible to produce offspring through artificial insemination (AI) by cryopreserving spermatozoa retrieved from the epididymis. Spermatozoa differ in maturation status along the epididymal duct, and only motile spermatozoa that have acquired fertilizing capacity are suitable for AI. Such spermatozoa can be collected from the terminal epididymal segment, located in the cauda epididymidis in the dog, and in both the corpus and cauda in the cat. Unlike ejaculated spermatozoa, epididymal spermatozoa have not been exposed to seminal plasma and therefore display distinct functional and structural characteristics. The method of sperm collection may also affect the sperm quality. While epididymal mincing results in the highest sperm numbers, it is associated with contamination of blood and epididymal tissue. Although numerous studies have reported successful cryopreservation of epididymal spermatozoa in dogs and cats, reports of live offspring following AI with frozen–thawed epididymal spermatozoa remain scarce. This review summarizes the physiological, anatomical, and functional distinctions between epididymal and ejaculated spermatozoa, emphasizing their implications for cryopreservation strategies and fertility outcomes.</p>