Inverted duplicates are a type of repetitive DNA motifs consist of two copies of reverse complementary sequences separated by a spacer sequence. They can lead to genome instability and many may have no function, but some functional small RNAs are processed from hairpins transcribed from these elements. It is not clear whether the pervasive numbers of such elements in genomes, especially those of mammals, is the result of high generation rates of neutral or slightly deleterious duplication events or positive selection for functionality. To test the functionality of intergenic inverted duplicates without known functions, we used mirror duplicates, a type of repetitive DNA motifs with few reported functions and little potential to form hairpins when transcribed, as a nonfunctional control. We identified large numbers of inverted duplicates within intergenic regions of human and mouse genomes, as well as 19 other vertebrate genomes. Structure characterization of these inverted duplicates revealed higher proportion to form stable hairpins compared with converted mirror duplicates, suggesting that inverted duplicates may produce hairpin RNAs. Expression profiling across tissues demonstrated that 7.8% of human and 5.7% of mouse inverted duplicates were expressed even under strict criteria. We found that expressed inverted duplicates were more likely to be structurally stable than both unexpressed inverted duplicates and expressed converted mirror duplicates. By dating inverted duplicates in the vertebrate phylogenetic tree, we observed higher conservation of inverted duplicates than mirror duplicates. These observations support the notion that expressed inverted duplicates may be functional through forming hairpin RNAs.