The first plantings were made in the spring of 1926. Because genetic diversity is the raw material for breeding, one of the first concerns was to assemble as many pine species and varieties as possible. The seeds used to establish the Arboretum came almost exclusively from native stands, whether from the United States or other countries, and their precise origins were meticulously recorded, often mapping the location of each parent tree. The arboretum was laid out in such a way that the species of pines were separated according to relationship groups established by Shaw in 1914 (The Genus Pinus). The Eddy Arboretum contains one of the best-documented collection of pines in the world, in addition to many other native and exotic conifers. Seventy-eight pine species, 24 firs, and many other conifers are presently included in the collection. Some sections of the Arboretum are devoted to collections that sample the entire range of genetic diversity in particular California conifers, such as Pinus lambertiana and Pinus coulterii.

The first hybrid pines ever produced by controlled pollination are in the Arboretum, and the collection includes crosses planted over 50 years ago. Over 90 hybrid combinations of pine species are represented, and often their backcrosses and second-generation offspring as well. These are the most valuable resources in the collection. Because of the long generation time of trees, the difficulties of working in their crowns, and even the long interval (18 months or more) between pollination and seed maturation makes, it unlikely that such a resource will ever be produced again. To duplicate some of these materials would require a research plan requiring over 40 years to execute. The Arboretum is a virtually irreplaceable genetic resource for tree breeders in California and beyond. The trees grown here provide living materials vital to studies of genetic variation.