Identifying an integrative framework that could appropriately delineate underlying mechanisms and individual risk/protective factors for human health has remained elusive. Evolutionary mismatch theory provides a comprehensive, integrative model for understanding the underlying causes and mechanisms of a wide range of modern health and well-being problems, ranging from obesity to depression. Despite growing interest regarding its importance though, no psychometrically-sound measure of evolutionary mismatch yet exists to facilitate research and intervention. To construct such a scale, aimed at gauging individual differences in the extent to which people's modern lifestyles are mismatched with ancestral conditions, we conducted four studies (a pilot study, followed by 3 main studies, with a final sample of 1901 participants across the main studies). Results from exploratory and confirmatory factor analyses have produced a 36-item evolutionary mismatched lifestyle scale (EMLS) with 7 subdomains of mismatched behaviours (e.g., diet, physical activity, relationships, social media use) that is psychometrically sound. Further, the EMLS is associated with physical, mental and subjective health. We explore the potential of the EMLS as a tool for examining interpersonal and cultural variations in health and wellbeing, while also discussing the limitations of the scale and future directions in relation to further psychometric examinations.