There are almost 1,700 species of mammals occurring in the Neotropical region broadly defined as including Mexico, Central America, the Caribbean Islands, and South America. The Barcode of Life Data (BOLD) Systems established an institutional collaboration with the Royal Ontario Museum (ROM) beginning in 2005 to DNA barcode the mammal tissue collections for the cytochrome oxidase subunit I (COI) gene region. To date, over 14,500 vouchered specimens have been sequenced representing nearly 19% (317 species) of the mammalian diversity in the Neotropics. This includes 11 of the 15 orders found in the Neotropics with the 4 missing orders representing less than 20 species. Almost half of the bat species (167 of 345) have been barcoded but only 3% (4 of 129) of the primate species have been done. Rodents are the most diverse order in the Neotropics, of which 12% of the species have been barcoded (106 of 898). In terms of geographic coverage, Middle America is the most comprehensively represented with only one country missing (Honduras), but half of the South American countries are not represented and there are no samples from the Caribbean. Although an initial DNA barcode reference library for Neotropical mammals has been established, there are obvious taxonomic and geographic areas that need to be surveyed to make it broadly applicable for species identification and discovery. Nevertheless, preliminary results indicate an underestimation of approximately one-third of the currently recognized species of mammals in the Neotropics, affirming it as a region of high biodiversity.