Transposable elements make up a significant fraction of many eukaryotic genomes. Although both classes of transposable elements, the DNA transposons and the retrotransposons, show substantial expansion in plants and invertebrates, the DNA transposons are thought to have become inactive in mammalian genomes long ago. Here, we report the first evidence for recent activity of DNA transposons in a mammalian lineage, the bat genus Myotis. Six recently active families of nonautonomous hobo/Activator/TAM transposons were identified in the Myotis lucifugus genome using computational tools. Low sequence divergence among the individual sequences and between individual sequences and their respective consensus sequences suggest their recent expansion in the M. lucifugus genome. Furthermore, amplification and sequencing of polymorphic insertion loci in a related taxon, M. austroriparius, confirms their recent activity. Myotis is one of the largest mammalian genera with 103 species. The discovery of DNA transposon activity in this genus may therefore influence our understanding of genome evolution and diversification in bats and in mammals in general. Furthermore, the identification of a likely autonomous element may lead to new approaches for mammalian genetic manipulation.