Needs for objective and automatic assessment of animal welfare are increasing in livestock production. The strong dam-offspring relationship is a good model to investigate the vocal expression of emotions in animals since removing the lamb induces behavioural distress in ewes (e.g. agitation and vocalisations). We analysed acoustic characteristics in bleats of ewes following an unpredictable separation from their lambs, in addition to their behavioural and physiological responses. Twenty four ewes of INRA 401 breed were individually exposed to 3 successive 3 min phases: 1) in contact with their lamb placed behind a grid, 2) at 6m away from the lamb and 3) again in contact. Behaviour, vocalisations and cardiac activity (via adhesive external electrodes and a telemetric remote system) were recorded and blood was collected just after the test by venipuncture for cortisol assay. After the lamb was moved away, the ewes were more active and more vigilant, they also bleated more, and their heart rate and cortisol levels dramatically increased. These results confirm that the separation induced behavioural and physiological responses of distress in ewes which reflect negative emotional states. Beside, acoustic analyses show that lamb withdrawal induced changes in the voice characteristics of mothers in correlation with their physiological and behavioural responses. Temporal parameters (e.g. total duration) increased, as did amplitude parameters (e.g. energy, RMS). Concerning the frequency parameters, fundamental frequency increased but frequency Bandwidth, Quartiles (Q25%), (Q50%), (Q75%) decreased. Therefore some bleat characteristics can be used as acoustic markers of emotional distress. Acoustic sensory modality could thus provide an objective basis of negative emotions in sheep, which measure could be easier to automate than other behavioural and physiological correlates of emotional reactions.