Epistasis is an important feature of the genetic architecture of quantitative traits. Previously, we showed that startle-induced locomotor behaviour of Drosophila melanogaster, a critical survival trait, is highly polygenic and exhibits epistasis. Here, we characterize epistatic interactions among homozygous P-element mutations affecting startle-induced locomotion in the Canton-S isogenic background and in 21 wild-derived inbred genetic backgrounds. We find pervasive epistasis for pairwise combinations of homozygous P-element insertional mutations as well as for mutations in wild-derived backgrounds. In all cases, the direction of the epistatic effects is to suppress the mutant phenotypes. The magnitude of the epistatic interactions in wild-derived backgrounds is highly correlated with the magnitude of the main effects of mutations, leading to phenotypic robustness of the startle response in the face of deleterious mutations. There is variation in the magnitude of epistasis among the wild-derived genetic backgrounds, indicating evolutionary potential for enhancing or suppressing effects of single mutations. These results provide a partial glimpse of the complex genetic network underlying the genetic architecture of startle behaviour and provide empirical support for the hypothesis that suppressing epistasis is the mechanism underlying genetic canalization of traits under strong stabilizing selection. Widespread suppressing epistasis will lead to underestimates of the main effects of quantitative trait loci (QTLs) in mapping experiments when not explicitly accounted for. In addition, suppressing epistasis could lead to underestimates of mutational variation for quantitative traits and overestimates of the strength of stabilizing selection, which has implications for maintenance of variation of complex traits by mutation–selection balance.