Attentional biases towards threatening stimuli are increased when animals are in anxious states. An ability to assess negatively valanced affective states such as anxiety are important as they may be related to poor welfare. Flight speed and crush score are two well established tests of temperament in cattle, but little is known about their relationship with anxiety. This study was conducted to validate an attention bias test as a measure of anxious states in cattle and to determine whether the temperament trait(s) measured by flight speed and crush score tests were associated with anxiety. Thirty six Angus steers received one of the following pharmacological treatments: 1) anxiogenic (increase anxiety; 1-methy-chlorophenylpiperazine; m-CPP), 2) anxiolytic (decrease anxiety; diazepam) or, 3) control (saline). Thirty minutes later the steers were tested for crush score, flight speed and attention bias. The attention bias test involved measuring the response of each animal to the presence of the threat of a dog for 10 s after which behavioural responses were recorded for 3 min. Steers treated with m-CPP showed increased attention towards the threat (P < 0.01), increased vigilance (P < 0.01), spent more time with their ears backwards (P = 0.022) and had a greater core temperature increase over the test period (P = 0.01) compared to the control and diazepam groups. Cattle treated with m-CPP were significantly slower and less likely to eat in the attention bias test, compared to control and diazepam treated groups (P = 0.04). The diazepam treatment did not affect any of the measures in the attention bias test (P > 0.05). Crush score and flight speed were not affected by treatment (P > 0.05). The results suggest that the pharmacologically induced anxiety state may not be related to these traditional tests of temperament. Increased attention towards the threat and differences in behaviours and body temperature during the attention bias test confirm that this test indicates increased anxiety in cattle. Attention bias testing provides promise as a measure of some negatively valanced affective states in cattle.