The U.S. National Entomological Collection ranks as the second largest insect collection in the world with approximately 35 million specimens including over 100,000 holotypes plus hundreds of thousands of additional paratypes and other secondary types.

The collection includes over 300,000 species representing approximately 60% of known insect families. With specimens from locations worldwide, the collections are second to none in coverage for the Nearctic and Neotropical regions. Specimens from the Old World are also well represented, especially from Sri Lanka, the Philippines, and Papua New Guinea. Particular strengths include mosquitoes, wasps, beetles, butterflies and moths, and flies. Although the bulk of the collection is kept dry, various groups—such as spiders—are stored in alcohol.

The collections are typically arranged by taxon; lower categories (genus, species) are arranged alphabetically, and for select taxa, they are further organized by country of origin within each species.

While the majority of the collection is housed at the National Museum of Natural History in Washington DC, some groups are held at other research facilities in nearby Maryland, including the USDA's Agricutural Research Center in Beltsville, Maryland and the Smithsonian Institution's Museum Support Center in Suitland, Maryland.

The collections at the Agricultural Research Center include the Acari (mites), the Sternorrhyncha (aphids, whiteflies, scale insects and jumping plant lice), and one of the largest Isoptera (termites) collections in the world.

The collections at the Museum Support Center constitute roughly 20% of insect holdings. Some of the groups stored entirely at MSC are the mosquitoes and other biting flies, crane flies, lice, fleas, ichneumonid wasps, mantids and walkingsticks, saturniid and sphingid moths, and papilionid butterflies.