ABSTRACT: Gammaherpesviruses (GHVs) are a diverse and rapidly expanding group of viruses associated with a variety of disease conditions in humans and animals. To identify felid GHVs, we screened domestic cat (Felis catus), bobcat (Lynx rufus), and puma (Puma concolor) blood cell DNA samples from California, Colorado, and Florida using a degenerate pan-GHV PCR. Additional pan-GHV and long-distance PCRs were used to sequence a contiguous 3.4-kb region of each putative virus species, including partial glycoprotein B and DNA polymerase genes. We identified three novel GHVs, each present predominantly in one felid species: Felis catus GHV 1 (FcaGHV1) in domestic cats, Lynx rufus GHV 1 (LruGHV1) in bobcats, and Puma concolor GHV 1 (PcoGHV1) in pumas. To estimate infection prevalence, we developed real-time quantitative PCR assays for each virus and screened additional DNA samples from all three species (n = 282). FcaGHV1 was detected in 16% of domestic cats across all study sites. LruGHV1 was detected in 47% of bobcats and 13% of pumas across all study sites, suggesting relatively common interspecific transmission. PcoGHV1 was detected in 6% of pumas, all from a specific region of Southern California. The risk of infection for each host varied with geographic location. Age was a positive risk factor for bobcat LruGHV1 infection, and age and being male were risk factors for domestic cat FcaGHV1 infection. Further characterization of these viruses may have significant health implications for domestic cats and may aid studies of free-ranging felid ecology.Gammaherpesviruses (GHVs) establish lifelong infection in many animal species and can cause cancer and other diseases in humans and animals. In this study, we identified the DNA sequences of three GHVs present in the blood of domestic cats (Felis catus), bobcats (Lynx rufus), and pumas (Puma concolor; also known as mountain lions, cougars, and panthers). We found that these viruses were closely related to, but distinct from, other known GHVs of animals and represent the first GHVs identified to be native to these feline species. We developed techniques to rapidly and specifically detect the DNA of these viruses in feline blood and found that the domestic cat and bobcat viruses were widespread across the United States. In contrast, puma virus was found only in a specific region of Southern California. Surprisingly, the bobcat virus was also detected in some pumas, suggesting relatively common virus transmission between these species. Adult domestic cats and bobcats were at greater risk for infection than juveniles. Male domestic cats were at greater risk for infection than females. This study identifies three new viruses that are widespread in three feline species, indicates risk factors for infection that may relate to the route of infection, and demonstrates cross-species transmission between bobcats and pumas. These newly identified viruses may have important effects on feline health and ecology.