Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution.Most DNA transposons belong to the cut-and-paste types,which are relatively simple elements characterized by terminal inverted repeats (TIRs) flanking a single gene encoding a transposase.All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion.Here,we report a new group of evolutionarily related DNA transposons called Spy,which also include TIRs and DDE motif-containing transposase,but surprisingly do not create TSDs upon insertion.Instead,Spy transposons appear to transpose precisely between 5’-AAA and TTT-3’ host nucleotides,without duplication or modification of the AAATTT target sites.Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches.Phylogenetic analyses indicate that Spy transposases are distantly related to IS5,ISL2EU and PIF/Harbinger transposases.However,Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference.Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties.