Novel members of the subfamily Gammaherpesvirinae, hosted by eight mammalian species from six orders (Primates, Artiodactyla, Perissodactyla, Carnivora, Scandentia, and Eulipotyphla), were discovered using PCR with pan-herpesvirus DNA polymerase (DPOL) gene primers and genus-specific glycoprotein B (gB) gene primers. The gB and DPOL sequences of each virus species were connected by long-distance PCR, and contiguous sequences of approximately 3.4 kbp were compiled. Six additional gammaherpesviruses from four mammalian host orders (Artiodactyla, Perissodactyla, Primates, and Proboscidea), for which only short DPOL sequences were known, were analyzed in the same manner. Together with available corresponding sequences for 31 other gammaherpesviruses, alignments of encoded amino acid sequences were made and used for phylogenetic analyses by maximum-likelihood and Bayesian Monte Carlo Markov chain methods to derive a tree which contained two major loci of unresolved branching details. The tree was rooted by parallel analyses that included alpha- and betaherpesvirus sequences. This gammaherpesvirus tree contains 11 major lineages and presents the widest view to date of phylogenetic relationships in any subfamily of the Herpesviridae, as well as the most complex in the number of deep lineages. The tree's branching pattern can be interpreted only in part in terms of the cospeciation of virus and host lineages, and a substantial incidence of the interspecies transfer of viruses must also be invoked.