Objective and issue-neutral qualitative assessments of livestock behavior could provide a powerful assessment of welfare, augmenting quantitative measures such as autonomic and endocrine changes, which are often difficult to assess under many commercial livestock conditions. We set out to validate the use of qualitative behavioral assessment (QBA) in sheep using controlled experimental conditions (transport as a challenge) and comparing assessments against physiological variables. The behavioral expression of 14 Merino wethers, which had never experienced land transport, were assessed during their first road event (naïve to transport), and then again on their seventh event, 8 d later (habituated to transport). Blood samples were collected immediately before loading and after unloading, and heart rate and core body temperature were measured continuously throughout each event. Continuous video footage recorded during each event was used to provide clips of individual animals that were shown to observers for QBA. There was significant consensus (P < 0.001) amongst 63 observers in terms of their assessment of the behavioral expression of the sheep. Transport-naïve sheep were assessed as being more ‘alert’, ‘anxious’, and ‘aware’, whereas transport-habituated sheep were more ‘comfortable’, ‘tired’, and ‘confident’ (P = 0.015). Heart rate and heart rate variability, core body temperature and a stress leukogram were greater (P < 0.05) in sheep during the first (naïve) event compared with the habituated event, and were significantly correlated with the QBA scores (P < 0.05). In conclusion, QBA is a valid, practical and informative measure of behavioral responses to transport.