Abstract The assumption that pleiotropic mutations are more deleterious than mutations with more restricted phenotypic effects is an important premise in models of evolution. However, empirical evidence supporting this assumption is limited. Here, we estimated the strength of stabilizing selection on mutations affecting gene expression in male Drosophila serrata. We estimated the mutational variance (VM) and the standing genetic variance (VG) from two well-matched panels of inbred lines: a panel of mutation accumulation (MA) lines derived from a single inbred ancestral line and a panel of inbred lines derived from an outbred population. For 855 gene-expression traits, we estimated the strength of stabilizing selection as s = VM/VG. Selection was observed to be relatively strong, with 17% of traits having s > 0.02, a magnitude typically associated with life-history traits. Randomly assigning expression traits to five-trait sets, we used factor analytic mixed modeling in the MA data set to identify covarying traits that shared pleiotropic mutations. By assigning traits to the same trait sets in the outbred line data set, we then estimated s for the combination of traits affected by pleiotropic mutation. For these pleiotropic combinations, the median s was three times greater than s acting on the individual component traits, and 46% of the pleiotropic trait combinations had s > 0.02. Although our analytical approach was biased toward detecting mutations with relatively large effects, likely overestimating the average strength of selection, our results provide widespread support for the prediction that stronger selection can act against mutations with pleiotropic effects.