Cut-and-paste DNA transposable elements are major components of eukaryotic genomes and are grouped into superfamilies (e.g., hAT, P) based on sequence similarity of the element-encoded transposase. The transposases from several superfamilies possess a protein domain containing an acidic amino acid triad (DDE or DDD) that catalyzes the "cut and paste" transposition reaction. However, it was unclear whether this domain was shared by the transposases from all superfamilies. Through multiple-alignment of transposase sequences from a diverse collection of previously identified and recently annotated elements from a wide range of organisms, we identified the putative DDE/D triad for all superfamilies. Furthermore, we identified additional highly conserved amino acid residues or motifs within the DDE/D domain that together form a "signature string" that is specific to each superfamily. These conserved residues or motifs were exploited as phylogenetic characters to infer evolutionary relationships among all superfamilies. The phylogenetic analysis revealed three major groups that were not previously discerned and led us to revise the classification of several currently recognized superfamilies. Taking the data together, this study suggests that all eukaryotic cut-and-paste transposable element superfamilies have a common evolutionary origin and establishes a phylogenetic framework for all future cut-and-paste transposase comparisons.