Several studies have evaluated the effects of excessive heat stress on chromatin damage; however, DNA damage due to ambient seasonal heat stress has yet to be evaluated. The objective of the present study was to evaluate the effects of natural heat stress on equine sperm chromatin structure by means of two tests: the sperm chromatin structure assay (SCSA) and Sperm-Halomax kit (Halo). Stallion semen samples were collected after a period of excessive heat load (31.05°C) and were compared with samples collected at lower ambient temperatures (14.26°C) to evaluate chromatin damage caused by ambient heat stress produced in the testes. The data indicate that there is no correlation between the SCSA and Halo assays (r = 0.18 and F = 0.48). Additionally, no correlation for ambient heat stress was observed for any of the applied techniques—SCSA, Halo, and live:dead staining (P =.2682,.4628, and.0377, respectively). These results suggest that ambient heat load has little effect on stallion chromatin damage. Other elements, such as relative humidity and heat index, were outside the realm of this study and should be assessed in future studies with a larger sample size.