Genetic differentiation in thermal adaptation can result from a trade-off between the performance of organisms across different temperatures or from the accumulation of deleterious mutations. In this experiment, we assayed thermal sensitivity of 138 genetically distinct Mycosphaerella graminicola isolates sampled from five host populations in four locations under two temperature regimes (22 and 15 °C) and found significant differences in growth rate and response to temperature among populations. On average, genetic differentiation accounted for more than 50% of phenotypic variation in thermal adaptation while plasticity contributed less than a quarter of phenotypic variation. Populations originating from warm places performed better under the high-temperature regime and had a larger positive response to increasing temperature. Pairwise population differentiation (Q(ST) ) in temperature sensitivity, measured by taking the ratio of growth rates at 22 to 15 °C, was positively and significantly correlated to the pairwise difference in annual mean temperature at the collection sites. Because overall Q(ST) in temperature sensitivity was significantly higher than overall G(ST) in neutral restriction fragment length polymorphism loci, we believe that the primary mechanism underlying this thermal adaptation is antagonistic pleiotropy. Our results indicate that temperature sensitivity is a better indicator of thermal adaptation than growth rate at individual temperatures.