Introduction and objectives The purpose of this study was to use high accurate mass metabolomic profiling to investigate differences within a phenotypically diverse canine population, with breed-related morphological, physiological and behavioural differences. Previously, using a broad metabolite fingerprinting approach, lipids appear to dominate inter-and intra-breed discrimination. The purpose here was to use Ultra High Performance Liquid Chromatography- High Resolution Mass Spectrometry (UHPLC-HRMS) to identify in more detail, inter-breed signatures in plasma lipidomic profiles of home-based, client-owned dogs maintained on different diets and fed according to their owners' feeding regimens. Methods Nine dog breeds were recruited in this study (Beagle, Chihuahua, Cocker Spaniel, Dachshund, Golden Retriever, Greyhound, German Shepherd, Labrador Retriever and Maltese: 7-12 dogs per breed). Metabolite profiling on a MTBE lipid extract of fasted plasma was performed using UHPLC-HRMS. Results Multivariate modelling and classification indicated that the main source of lipidome variance was between the three breeds Chihuahua, Dachshund and Greyhound and the other six breeds, however some intrabreed variance was evident in Labrador Retrievers. Metabolites associated with dietary intake impacted on breed-associated variance and following filtering of these signals out of the data-set unique inter-breed lipidome differences for Chihuahua, Golden Retriever and Greyhound were identified. Conclusion By using a phenotypically diverse home-based canine population, we were able to show that high accurate mass lipidomics can enable identification of metabolites in the first pass plasma profile, capturing distinct metabolomic variability associated with genetic differences, despite environmental and dietary variability.