The emotional valence hypothesis suggests an increased left-brain hemisphere activation during positive situations and vice versa during negative situations. Since facial thermal asymmetries may reflect lateralised brain activity, we investigated this in dairy calves subjected to hot-iron disbudding (Disbudded; n = 12) as a model of negative affective states. As affective responses can vary due to previous experiences, we examined whether calves that had (ExpObs; n = 12) and had not (InexObs; n = 12) experienced disbudding differed in their thermal response to a conspecific being disbudded, and whether calf response to the researcher (approaching, moving away, not moving) was associated with thermal asymmetries. We made thermographic recordings of each calf on three days: Day before disbudding (D1); Disbudding day (D2); and Day after disbudding (D3), and at two different times: Disbudding time/1400(T1) and Afternoon/1700(T2). Data were analysed using multilevel models. Calves had warmer left ears on D2 compared to higher temperatures on the right ear on D1, suggesting higher right-hemisphere activity on D2. ExpObs calves had higher left-eye temperatures when observing a conspecific being disbudded (D2×T1) than InexObs calves that had warmer right eyes, but this reversed on the following day (D3×T1). Calves avoiding the researcher had warmer left eyes whereas those approaching him had warmer right eyes, suggesting greater activation of the right hemisphere in the former. This study provides initial evidence of temperature asymmetries when observing or experiencing a negative event. Further work is required to confirm and build upon these early findings. The study highlights the potential for future development of methods using infrared thermography as a proxy measure of affective valence.