The in vitro function of sex-sorted, frozen–thawed ram spermatozoa derived from fresh or frozen semen was investigated. Sorted, frozen–thawed spermatozoa had higher (P < 0.05) motility, viability, acrosome integrity and mitochondrial activity than non-sorted, frozen–thawed controls immediately following thawing and after incubation at 37°C for 3 and 6 h. Similarly, frozen–thawed, sorted, re-frozen–thawed spermatozoa outperformed (P < 0.05) non-sorted controls upon thawing (mitochondrial activity) and following a 3-h incubation (motility, viability/acrosome integrity and mitochondrial activity), but there were no differences after incubation for 6 h (P > 0.05). Velocity characteristics (computer assisted sperm assessment 0–6 h post-thaw) of sorted spermatozoa derived from either fresh or frozen semen remained inferior (P < 0.05) to non-sorted spermatozoa, as did their ability to penetrate artificial cervical mucus after thawing. Direct comparison of cryopreserved spermatozoa derived from either fresh or frozen semen revealed that frozen–thawed, sorted, re-frozen–thawed spermatozoa had comparable (P > 0.05) motility, viability/acrosome integrity, mitochondrial activity, average path velocity and oviducal binding capacity immediately post-thaw, but reduced (P < 0.05) quality after 3 and 6 h of incubation. These findings indicate that, under the tested in vitro conditions, sex-sorted spermatozoa derived from fresh semen are superior in some respects to those derived from frozen semen. Further, that the use of either technique, while reducing velocity characteristics and cervical mucus penetration, results in comparable, if not enhanced motility, membrane and mitochondrial function in the post-thaw population of spermatozoa when compared with non-sorted, frozen–thawed controls.