Background: The skin barrier is important in the pathogenesis of atopic dermatitis and stratum corneum lipids have a critical role. Skin surface lipids have been largely overlooked but also contribute to barrier function. An untargeted approach was used to compare the skin surface lipids from atopic and non-atopic West Highland White terrier dogs (WHWT). Objectives: The primary hypothesis was that a difference in the lipidome would exist. The secondary hypothesis was that affected and unaffected skin lipids would differ. Animals and methods: This prospective, cross-sectional, case-controlled study included thirty-nine privately owned WHWTs. Dogs were assigned to one of four disease status groups based on strict criteria. Samples for lipid analysis were collected from the skin surface of unaffected and affected sites. Lipid analysis was by untargeted liquid chromatography/mass spectrometry and utilised lipid identification software packages. Principle component analysis (PCA) and partial least-squares discriminant analysis (sPLS-DA) statistical methods analysed the association between the relative lipid abundance and disease status and affected and unaffected skin. Results: Samples for lipid analysis found 421 lipid soluble features of which ten lipids were positively identified. Statistical analysis could not distinguish between non-atopic and atopic dog but did reveal a statistically significant difference in the lipid profiles from affected and non-affected skin irrespective of disease status. Conclusions: A large array of unidentified lipids from the skin surface were found with a difference between affected and unaffected skin unrelated to disease status. Investigation into the lipidome of the skin surface is an emerging area of research with clinical and therapeutic applications.