Feral swine (Sus scrofa) are a pathogen reservoir for pseudorabies virus (PrV). The virus can be fatal to wildlife and contributes to economic losses in the swine industry worldwide. National surveillance efforts in the US use serology to detect PrV-specific antibodies in feral swine populations, but PrV exposure is not a direct indicator of pathogen transmission among conspecifics or to non-suid wildlife species. We measured antibody production and the presence of PrV DNA in four tissue types from feral swine populations of Florida, US. We sampled blood, nasal, oral, and genital swabs from 551 individuals at 39 sites during 2014–16. Of the animals tested for antibody production, 224 of 436 (51%) feral swine were antibody positive while 38 of 549 feral swine (7%) tested for viral shedding were quantitative polymerase chain reaction (qPCR)-positive for PrV. The detection of PrV DNA across all the collected sample types (blood, nasal, oral, and genital [vaginal] swabs) suggested viral shedding via direct (oronasal or venereal), and potentially indirect (through carcass consumption), routes of transmission among infected and susceptible animals. Fourteen of 212 seronegative feral swine were qPCR-positive, indicating 7% false negatives in the serologic assay. Our findings suggest that serology may underestimate the actual infection risk posed by feral swine to other species and that feral swine populations in Florida are capable of shedding the virus through multiple routes.