In vertebrates, seven signal transducer and activator of transcription (STAT) proteins bind to palindromic sites separated by spacers of two or three nucleotides (STAT1), four nucleotides (STAT6) or three nucleotides (STAT2 to STAT5a/b). This diversity of binding sites provides specificity to counter semiredundancy and was thought to be a recent evolutionary acquisition. Here, we examine the natural DNA-binding sites of the single Drosophila Stat and show that this is not the case. Rather, Drosophila Stat92E is able to bind to and activate target gene expression through both 3n and 4n spaced sites. Our experiments indicate that Stat92E has a higher binding affinity for 3n sites than for 4n sites and suggest that the levels of target gene expression can be modulated by insertion and/or deletion of single bases. Our results indicate that the ancestral STAT protein had the capacity to bind to 3n and 4n sites and that specific STAT binding preferences evolved with the radiation of the vertebrate STAT family.