Negative emotional states in humans are associated with a negative (pessimistic) response bias towards ambiguous cues in judgement tasks. Every mammalian young is eventually weaned; this period of increasing nutritional and social independence from the dam is associated with a pronounced behavioural response, especially when weaning is abrupt as commonly occurs in farm animals. The aim of the current study was to test the effect of separation from the cow on the responses of dairy calves in a judgement task. Thirteen Holstein calves were reared with their dams and trained to discriminate between red and white colours displayed on a computer monitor. These colours predicted reward or punishment outcomes using a go/no-go task. A reward was provided when calves approached the white screen and calves were punished with a timeout when they approached the red screen. Calves were then tested with non-reinforced ambiguous probes (screen colours intermediate to the two training colours). "GO" responses to these probes averaged (± SE) 72±3.6% before separation but declined to 62±3.6% after separation from the dam. This bias was similar to that shown by calves experiencing pain in the hours after hot-iron dehorning. These results provide the first evidence of a pessimistic judgement bias in animals following maternal separation and are indicative of low mood.