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Abstract Population differentiation is a pervasive process in nature. At present, evolutionary studies on plant population differentiation address key questions by undertaking joint ecological and genetic approaches and employing a combination of molecular and experimental means. In this special issue, we gathered a collection of papers dealing with various ecological and genetic aspects of population differentiation in plants. In particular, this special issue encompasses eight research articles and two reviews covering a wide array of worldwide environments, plant functional types, genetic and genomic approaches, and common garden experiments to quantify molecular and/or quantitative trait differentiation in plant populations. Overall, this special issue stresses the validity of traditional evolutionary studies focused on plant populations, whilst emphasizing the integration of classical biological disciplines and state-of-the-art molecular techniques into a unique toolkit for evolutionary plant research.