The need for objective assessment of animal welfare in livestock production increases. The measurement of psycho-acoustic markers could offer a practical alternative to the current subjective 'measures' of animals mental well-being. The mother-offspring sheep relationship offers a good model to investigate emotions in animals because ewes show strong behavioural responses when separated from their lambs. The aim of the present work was to identify the psycho-acoustic markers of positive and negative emotions in sheep. We analysed the acoustic characteristics of bleats emitted by the mother when its lambs were kept 1m away and without contact with their mother (distressing situation), in ewes selected for their high (nervous, n=15) or low (calm, n=12) reactivity to social isolation. Sound recordings were conducted at 48hr after lambing. The modality and complexity of stress-encoding parameters were analysed using ANOVA and discriminant function analysis (DFA). Lamb withdrawal induced changes in the mother voice characteristics. Frequency parameters (e.g. Fundamental frequency (F0), 120.2±3.9 vs 133.3±3.5Hz) increased, as did temporal and amplitude parameters (e.g. total duration: 829±32 vs 984±34ms; RMS: 45.7±3.5 vs 66.9±4.2mv/unit; ANOVA, p<0.05). Six major acoustic markers differed from the calm to nervous selected ewes: F0 (+16%), max. F0 time (+16.5%), Root Mean Square (+28.7%) were higher for the nervous than the calm ewes and the depth of Amplitude-Modulation (+58.7%), Shimmer (+23%) and Jitter (+36%) were greater for calm than for nervous ones (ANOVA, p<0.05). Using DFA, almost 90% of the animals could be correctly assigned to one of the two lines using only these 6 major acoustic parameters (only 11% misclassified in each line when we inject new data). Our results suggest that some ewe bleat characteristics could be used as psycho-acoustic markers of emotions. This study also shows that acoustic sensory modality could provide an objective basis for assessing animal emotion.