Mycoplasma alligatoris causes acute lethal infection of alligators (Alligator mississippiensis). The objective of this study was to assess the current seroprevalence of M. alligatoris among free-ranging, juvenile and subadult alligators in Florida. Thirty-two of 592 (5.4%) plasma samples from alligators at 12 of 20 sites (60%) in April and October 2003 were tested seropositive (titer 1: ≥32) by enzyme-linked immunosorbent assay for anti–M. alligatoris antibodies. These results show that alligators throughout Florida have a recent history of exposure to M. alligatoris and suggest that contact with free-ranging alligators may constitute a risk of lethal infection of susceptible crocodilians.