Abstract: Observational field studies were conducted on two remote populations of feral horses in Australia to determine whether lateralization is a characteristic of Equus caballus as a species or results from handling by humans. Group 1 had been feral for two to five generations and Group 2 for 10–20 generations. In both groups, left-side biases were present during agonistic interactions and in reactivity and vigilance. Therefore, as in other vertebrates, the right hemisphere appears to be specialized to control agonistic behaviour and responses to potential threats. The leftwards bias was stronger in measures of behaviour involving more aggression and reactivity. Preferences to place one forelimb in front of the other during grazing were also determined. No population bias of forelimb preference was found, suggesting that such limb preferences present in domestic horses may be entrained. Since stronger individual limb preferences were found in immature than in adult feral horses, limb preference may be modified by maturation or experience in the natural habitat. Stronger limb preference was associated significantly with elevated attention to the environment but only in younger feral horses. No sex differences in lateralization were found. The findings are evidence that horses show visual lateralization, as in other vertebrates, not dependent on handling by humans. Limb preference during grazing, by contrast, does appear to depend on experience.