The domestic cat is now one of the most common pet species in the Western world. As part of its role as a pet, cats are expected to not only tolerate but enjoy being touched. This study consisted of two experiments, with the first investigating the influence of body region touched and handler familiarity on the domestic cat's behavioural response to being stroked. The second experiment extended this work by investigating the influence of order of body region touched on behavioural responses. Both handler familiarity and body region stroked significantly influenced negative behavioural responses. Familiar handling, in comparison to unfamiliar handling, led to significantly higher negative behavioural scores displayed by the cats (score of 8.5 versus 5.0 respectively, p = 0.001). When considering the different body regions investigated, the caudal region produced the highest negative scores both when handled by the unfamiliar person (Experiment 1: score of 2.0 in comparison to scores of 0.0–1.0 for other studied body regions, p = 0.046) and by the familiar person (Experiment 2: score of 2.0 in comparison to a score of 1.0 for each other studied body region, p = 0.002). Order of body region touched had no significant bearing on behavioural responses exhibited. Results suggest that handling of cats should avoid the caudal region and highlight the need for further investigation into the owner–cat relationship.