Environmental and biochemical factors can compromise the DNA integrity of equine spermatozoa. Several sperm DNA damage assays exist, but their relative prognostic value has not been assessed in the horse. This study compared four assays for detecting DNA damage in stallion spermatozoa: the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion assay (SCD), the single-cell gel electrophoresis (comet) assay, and the 8-hydroxy-2′-deoxyguanosine (8-OHdG) ELISA. Experiment 1 assessed the effect of hydrogen peroxide (H₂O₂), a driver of oxidative stress, on sperm DNA damage across increasing concentrations and incubation times. Experiment 2 assessed arachidonic acid (AA), an upstream driver of endogenous oxidative stress. Experiment 3 evaluated the prognostic value of each assay using semen from Thoroughbred stallions during periods of high and low fertility. Across experiments, the comet assay detected DNA damage at lower stressor doses and shorter incubation times than the other assays and was the only assay to distinguish semen from low-fertility stallions from that of high-fertility stallions. The SCSA, SCD, and 8-OHdG ELISA were comparatively less sensitive in detecting significant differences under the same conditions. In conclusion, the comet assay demonstrated the most consistent responsiveness in detecting sperm DNA damage in stallions under the tested conditions.