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During the past decade, microevolution of intermediary metabolism has become an important new research focus at the interface between metabolic biochemistry and evolutionary genetics. Increasing recognition of the importance of integrative studies in evolutionary analysis, the rising interest in ‘evolutionary systems biology’, and the development of various ‘omics’ technologies have all contributed significantly to this developing interface. The present review primarily focuses on five prominent areas of recent research on pathway microevolution: lipid metabolism and life-history evolution; the electron transport system, hybrid breakdown and speciation; glycolysis, alcohol metabolism and population adaptation in Drosophila; chemostat selection in microorganisms; and anthocyanin pigment biosynthesis and flower color evolution. Some of these studies have provided a new perspective on important evolutionary topics that have not been investigated extensively from a biochemical perspective (hybrid breakdown, parallel evolution). Other studies have provided new data that augment previous biochemical information, resulting in a deeper understanding of evolutionary mechanisms (allozymes and biochemical adaptation to climate, life-history evolution, flower pigments and the genetics of adaptation). Finally, other studies have provided new insights into how the function or position of an enzyme in a pathway influences its evolutionary dynamics, in addition to providing powerful experimental models for investigations of network evolution. Microevolutionary studies of metabolic pathways will undoubtedly become increasingly important in the future because of the central importance of intermediary metabolism in organismal fitness, the wealth of biochemical data being provided by various omics technologies, and the increasing influence of integrative and systems perspectives in biology.