In the last few centuries, the domestic dog has undergone selection for specific traits and breed diversification, forming genetically isolated populations. It is evident that certain breeds are predisposed to simple Mendelian disorders and complex diseases such as cancer. Phenotypic variation in metabolite levels in plasma will vary with genotype, and may reflect susceptibility to disease. Therefore, analysis of the source and extent of variation will potentially be useful in disease investigation and biomarker development. The aim of this study was to characterise the natural metabolite variation and identify genes involved in regulating metabolite abundance in healthy German Shepherd dogs. Using the Illumina Canine HD array, 82 healthy German Shepherd dogs were genotyped across over 172,000 markers. Plasma metabolites, including amino acids, sugars, and fatty acids, of these dogs were quantified using targeted liquid chromatography-mass spectrometry. Genome-wide association analyses of plasma metabolites were conducted to examine genetic influences on metabolite variation.