Mutational hotspots are common in disease genes and in complex genomic regions not amenable to sequencing by short reads technologies. However, it is still not clear what makes a particular genomic region more prone to mutations and whether the different mutations located in a hotspot are functionally equivalent. In this work, we have discovered and characterized in detail an insertional hotspot in the promoter region of a stress-response gene in the fruitfly Drosophila melanogaster. The nine transposable elements insertions described are clustered in a small 368 bp region and all belong to the same family of transposable elements: the roo family. Although the sequences of these insertions are highly similar, their molecular and functional consequences are different: only FBti0019985 insertion is associated with increased resistance to cold-stress. Interestingly, the previously described insertional hotspot in the D. melanogaster genome was also located in the promoter of a stress response gene suggesting that selection may favour the maintenance of genetic variability in these genes.