The role of the YXFGLa family of allatostatin (AST) peptides in dipterans is not well-established. The recent completion of sequencing of genomes for multiple Drosophila species provides an opportunity to study the evolutionary variation of the allatostatins and to examine regulatory elements that control gene expression. We performed comparative analyses of Ast genes from seven Drosophila species (Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, Drosophila yakuba, Drosophila pseudoobscura, Drosophila mojavensis, and Drosophila grimshawi) and used phylogenetic footprinting methods to identify conserved noncoding motifs, which are candidates for regulatory regions. The peptides encoded by the Ast precursor are nearly identical across species with the exception of AST-1, in which the leading residue may be either methionine or valine. Phylogenetic footprinting predicts as few as 3, to as many as 17 potential regulatory sites depending on the parameters used during analysis. These include a Hunchback motif approximately 1.2 kb upstream of the open reading frame (ORF), overlapping motifs for two Broad-complex isoforms in the first intron, and a CF2-II motif located in the 3'-UTR. Understanding the regulatory elements involved in Ast expression may provide insight into the function of this neuropeptide family.