Isozymes are enzymes that differ in sequence but catalyze the same chemical reactions. Despite their apparent redundancy, isozymes are often retained over evolutionary time, suggesting that they contribute to fitness. We developed an unsupervised computational method for identifying environmental conditions under which isozymes are likely to make fitness contributions. This method analyzes published gene expression data to find specific experimental perturbations that induce differential isozyme expression. In yeast, we found that isozymes are strongly enriched in the pathways of central carbon metabolism and that many isozyme pairs show anticorrelated expression during the respirofermentative shift. Building on these observations, we assigned function to two minor central carbon isozymes, aconitase 2 (ACO2) and pyruvate kinase 2 (PYK2). ACO2 is expressed during fermentation and proves advantageous when glucose is limiting. PYK2 is expressed during respiration and proves advantageous for growth on three-carbon substrates. PYK2’s deletion can be rescued by expressing the major pyruvate kinase only if that enzyme carries mutations mirroring PYK2’s allosteric regulation. Thus, central carbon isozymes help to optimize allosteric metabolic regulation under a broad range of potential nutrient conditions while requiring only a small number of transcriptional states. IMPORTANCE Gene duplication is one of the main evolutionary paths to new protein function. Typically, duplicated genes either accumulate mutations and degrade into pseudogenes or are retained and diverge in function. Some duplicated genes, however, show long-term persistence without apparently acquiring new function. An important class of isozymes consists of those that catalyze the same reaction in the same compartment, where knockout of one isozyme causes no known functional defect. Here we present an approach to assigning specific functional roles to seemingly redundant isozymes. First, gene expression data are analyzed computationally to identify conditions under which isozyme expression diverges. Then, knockouts are compared under those conditions. This approach revealed that the expression of many yeast isozymes diverges in response to carbon availability and that carbon source manipulations can induce fitness phenotypes for seemingly redundant isozymes. A driver of these fitness phenotypes is differential allosteric enzyme regulation, indicating isozyme divergence to achieve more-optimal control of metabolism.