Up to five million pet dogs in the UK (∼50% of the population) may, at some stage of their lives, perform undesirable separation-related behaviour (SRB) when left home alone [1], including vocalising, destruction and toileting [2]. Some owners perceive their dog to be ‘fine’ or even ‘happy’ when performing SRB [3], a few seek professional help [1], and others relinquish the dog [4]. Given the magnitude of the issue and the varied perceptions and behaviour of owners, the underlying emotional (affective) states of dogs showing SRB, and hence their welfare, requires elucidation. Whilst most dogs are believed to be anxious when showing SRB [1,2], it is uncertain whether their background affective state (mood) when they are not separated is also negative [1]. Here we use a new ‘cognitive bias’ measure of animal affect to show that dogs which exhibit high levels of SRB in a separation test also appear to have a more negative underlying mood.