Vocal expression of emotions has been suggested to be conserved throughout evolution. However, since vocal indicators of emotions have never been compared between closely related species using similar methods, it remains unclear whether this is the case. Here, we investigated vocal indicators of emotional valence (negative versus positive) in Przewalski’s horses, in order to find out if expression of valence is similar between species and notably among Equidae through a comparison with previous results obtained in domestic horse whinnies. We observed Przewalski’s horses in naturally occurring contexts characterised by positive or negative valence. As emotional arousal (bodily activation) can act as a confounding factor in the search for indicators of valence, we controlled for its effect on vocal parameters using a behavioural indicator (movement). We found that positive and negative situations were associated with specific types of calls. Additionally, the acoustic structure of calls differed according to the valence. There were some similarities but also striking differences in expression of valence between Przewalski’s and domestic horses, suggesting that vocal expression of emotional valence, unlike emotional arousal, could be species specific rather than conserved throughout evolution.