Despite the importance of molecular phylogenetics, few of its assumptions have been tested with real data. It is commonly assumed that nonparametric bootstrap values are an underestimate of the actual support, Bayesian posterior probabilities are an overestimate of the actual support, and among-gene phylogenetic conflict is low. We directly tested these assumptions by using a well-supported yeast reference tree. We found that bootstrap values were not significantly different from accuracy. Bayesian support values were, however, significant overestimates of accuracy but still had low false-positive error rates (0% to 2.8%) at the highest values (>99%). Although we found evidence for a branch-length bias contributing to conflict, there was little evidence for widespread, strongly supported among-gene conflict from bootstraps. The results demonstrate that caution is warranted concerning conclusions of conflict based on the assumption of underestimation for support values in real data.