Theoretical studies have predicted a trade-off between growth rate and yield in heterotrophic organisms. Here we test for the existence of this trade-off by analyzing the growth characteristics of 12 E. coli B populations that evolved for 20,000 generations under a constant selection regime. We performed three different tests. First, we analyzed changes in growth rate and yield over evolutionary time for each population. Second, we tested for a negative correlation between rate and yield across the 12 populations. Finally, we isolated clones from four selected populations and tested for a negative correlation between rate and yield within these populations. We did not find evidence for a trade-off based on the first two tests. However, we did observe a trade-off based on the within-population correlation of yield and rate. Our results indicate that, at least for the populations studied here, an analysis of the within-population diversity might be the most sensitive test for the existence of a trade-off. The observation of a trade-off within, but not between, populations suggests that the populations evolved different genetic solutions for growth in the selective environment, which in turn led to different physiological constraints.