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Human milk provides essential substrates for the optimal growth and development of a breastfed infant. Besides providing nutrients to the infant, human milk also contains metabolites which form an intricate system between maternal lifestyle, such as the mother’s diet and the gut microbiome, and infant outcomes. This study investigates the variation of these human milk metabolites from five different countries. Human milk samples (n = 109) were collected one month postpartum from Australia, Japan, the USA, Norway, and South Africa and were analyzed by nuclear magnetic resonance. The partial least squares discriminant analysis (PLS-DA) showed separation between either maternal countries of origin or ethnicities. Variation between countries in concentration of metabolites, such as 2-oxoglutarate, creatine, and glutamine, in human milk, between countries, could provide insights into problems, such as mastitis and/or impaired functions of the mammary glands. Several important markers of milk production, such as lactose, betaine, creatine, glutamate, and glutamine, showed good correlation between each metabolite. This work highlights the importance of milk metabolites with respect to maternal lifestyle and the environment, and also provides the framework for future breastfeeding and microbiome studies in a global context.