Two separate populations of threatened gopher tortoises (Gopherus polyphemus) in Florida were found to carry Salmonella spp. during relocation efforts from Orange and St. Johns Counties to Walton County. The potentially zoonotic bacteria were detected using cloacal swabs for culture and PCR analysis. Although there was a difference in the amount of perceived clinical stress undergone in each group, the percentage of tortoises shedding Salmonella was not statistically significant between the cohorts (9% and 11%). All isolates underwent antibiotic sensitivity testing and were susceptible to 10 antibiotic compounds.