The aim of this study was to determine the seroprevalence and serotype-specificity of the circulating antibodies against Foot-and-Mouth Disease Virus (FMDV) in cattle in Kasese and Bushenyi districts in Uganda. A total of 309 serum samples were collected and tested for antibodies against Non-Structural (NS) and Structural Proteins (SP) using Ceditest® FMDV-NS and Ceditest® FMDV type O test kits. Seroprevalences were much higher in Kasese in both tests (61 and 43%, respectively) than in Bushenyi (3 and 4%, respectively). A high proportion of sera, that tested positive in the NSP test, were subjected to seven serotype specific blocking ELISAs for antibodies against the seven FMDV serotypes (O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3). The study showed presence of antibodies against four FMDV serotypes with decreasing magnitude as follows: O> SAT 1> SAT 3/SAT 2. It is recommended to develop sampling schemes to include virus recovery and identification, as well as to focus serum sampling on young unvaccinated stock.