Feline leukemia virus (FeLV) was not detected in Florida pumas (Puma concolor coryi) in almost 20 yr of surveillance; however, the finding of two FeLV antigen-positive pumas during the 2002–2003 capture season led to an investigation of FeLV in the population. Between January 1990 and April 2007, the proportion of pumas testing FeLV antibody positive increased, with antibody-positive pumas concentrated in the northern portion of puma range. Five of 131 (4%) pumas sampled between July 2000 and April 2007 were viremic, with all cases clustered in Okaloacoochee Slough (OKS). Clinical signs and clinical pathology at capture were absent or included lymphadenopathy, moderate-to-severe anemia, and lymphopenia. All viremic pumas died; causes of death were septicemia (n=2), intraspecific aggression (n=2), and anemia/dehydration (n=1). Outcome after FeLV exposure in pumas was similar to that in domestic cats, with evidence of regressive, latent, and persistent infections. Management of the epizootic included vaccination, and as of April 2007, 52 free-ranging pumas had received one or more inoculations. Vaccinations were concentrated in OKS and in a band between OKS and the remainder of the puma population. There have been no new cases since July 2004; however, the potential for reintroduction of the virus remains.