AbstractEcological diversification depends on the extent of genetic variation and on the pattern of covariation with respect to ecological opportunities. We investigated the pattern of utilization of carbon substrates in wild populations of budding yeast Saccharomyces paradoxus. All isolates grew well on a core diet of about 10 substrates, and most were also able to grow on a much larger ancillary diet comprising most of the 190 substrates we tested. There was substantial genetic variation within each population for some substrates. We found geographical variation of substrate use at continental, regional, and local scales. Isolates from Europe and North America could be distinguished on the basis of the pattern of yield across substrates. Two geographical races at the North American sites also differed in the pattern of substrate utilization. Substrate utilization patterns were also geographically correlated at local spatial scales. Pairwise genetic correlations between substrates were predominantly positive, reflecting overall variation in metabolic performance, but there was a consistent negative correlation between categories of substrates in two cases: between the core diet and the ancillary diet, and between pentose and hexose sugars. Such negative correlations in the utilization of substrate from different categories may indicate either intrinsic physiological trade‐offs for the uptake and utilization of substrates from different categories, or the accumulation of conditionally neutral mutations. Divergence in substrate use accompanies genetic divergence at all spatial scales in S. paradoxus and may contribute to race formation and speciation.