The influence of different extender osmolality levels and the presence of different cryoprotectants on the post-thawed semen's characteristics and post-thawed plasma membrane integrity of ram spermatozoa were studied. Ram semen was frozen with TRIS-egg yolk based extender according to two-step dilution procedures. The final concentrations of the cryoprotectants: 6% glycerol, 6% 1,2-propanediol, 62.5 mM sucrose, and 62.5 mM trehalose were studied in three different extender B osmolality levels (350, 375, and 400 mOsm). The osmolality affected significantly the post-thawed semen's motility, defected acrosomes (DA), total morphological defect (TMD), along with the sperm's plasma membrane integrity (HOST). Type of cryoprotectant exerted significant effect (P<0.001) on the post-thawed semen's motility, DA, TMD, and HOST. There was a significant interaction between the osmolality and cryoprotectant on the post-thawed motility, DA and TMD, but not on the HOST. In general, post-thawed motility, acrosomal, morphological, and membrane integrity of the semen frozen with semen extender at 400 mOsm were better than those of 350 and 375 mOsm, regardless of the type of cryoprotectant. Glycerol and 1,2-propanediol, compared to sucrose, trehalose, and control groups, did not protect the post-thawed acrosome and morphological integrity, though it did protect motility and HOST. It was concluded that glycerol based extenders with a high osmotic pressure (400 mOsm) was a better choice for ram semen freezing compared to sucrose, trehalose, and cryoprotectant free extenders. The detrimental effect of glycerol on DA and TMD could be overcome by combining glycerol with sugars and by increasing the osmotic pressure of the extender used for semen cryopreservation. Further research on the cryopreservation of ram semen should focus on the extender osmolality and combination of different cryoprotectants.