Locomotion is a primary means by which animals interact with the world. To understand the contribution of genotype × environment interactions to individual differences in D. melanogaster larval locomotion we investigated phenotypic sensitivity to environmental changes in four strains of this species and their F1 hybrids. We also investigated to what extent flexibility and plasticity of locomotion depend upon larval age. Specifically, we examined larval locomotion at 48 and 96 h of development on three different substrates. Locomotion was influenced by the structure of the substrate, but this depended on both the genotype and larval age. At 48 h of larval development phenotypic variation in locomotion was attributable to both genotype × environment interactions and genotypic differences among the larvae, while at 96 h of age, differences were mainly due to genotype × environment interactions. An analysis of variance of the 4 × 4 diallel cross made at 48 and 96 h of development showed, depending on the cross, either dominance to increase/decrease locomotion, overdominance to increase/decrease locomotion, or no dominance to increase/decrease locomotion. Furthermore, the diversity of behavioral responses in the F1 hybrids was greater at 96 than at 48 h of larval development. Our results demonstrate that genotype × environment interactions result in plasticity of D. melanogaster larval locomotion, which makes sense in light of the fact that larvae, in the wild, develop in heterogeneous and rapidly changing environments.