The present study aimed to investigate the effect of different concentrations of sodium dodecyl sulfate (SDS), egg yolk and glycerol on the freezability and DNA integrity of stallion spermatozoa. Semen samples were collected from 7 Arabian stallions, centrifuged, extended in INRA-82 supplemented with different concentrations of SDS (0.00, 0.01, 0.02, 0.03 and 0.04%), egg yolk (5, 10, 15 and 20%) and glycerol (3, 4, 5, 6 and 7%), and processesed for cryopreservation. Post-thaw motility, acrosome and membrane integrity of spermatozoa were assessed and comet assay were applied to determine DNA integrity of spermatozoa. Results showed that the addition of appropriate concentration of SDS is fundamental for equine semen cryopreservation. SDS, egg yolk and glycerol at concentrations of 0.03%, 15% and 5%, respectively, resulted in the optimum motility, acrosome, membrane and DNA integrities of equine spermatozoa. Increasing the concentration of glycerol above 5% appeared to have deleterious effect of sperm DNA manifested as increased in the percentage of fragmented DNA, DNA content in tail of comet, tail length and Olive tail moment.  In conclusion, 0.03% SDS, 15% egg yolk and 5% glycerol are the optimum concentrations for cryopreservation of equine spermatozoa, and comet assay is a valuable tool to monitor DNA cryo-damage in stallion sperm.