Foot-and-mouth disease (FMD) is endemic in Egypt and in most parts of Africa causing huge economic losses. Control of FMD using vaccination requires information on the occurrence of various FMDV serotypes. This study aimed to determine the prevalence of FMDV serotypes in Sharkia Governorate, Egypt. A total number of 643 different samples, within ten different localities, were collected from both cattle and buffaloes (n = 283) of different, age, sex, immune status against FMD, and health status. Field samples (n = 360) have been screened for FMDV by RT-PCR using universal primers and were further subtyped using serotype-specific primers. Additionally, serum samples (n = 283) have been analyzed by applying FMDV serotype-specific antibody ELISA. The RT-PCR screening revealed that a total number of 39/283 (13.8%), 61/283 (21.6%) and 17/38 (44.7%) animals were positive for FMDV serotype O, A and SAT2, respectively. While, by ELISA, neutralizing antibodies directed against FMDV serotype O, A, and SAT2, were found in 177/283 (62.5%), 171/283 (60.4%) and 27/38 (71.1%) serum samples, respectively. These results indicated the endemic status of the FMDV serotypes O, A and SAT2 in Sharkia Governorate despite routine FMD vaccination programs. Although many variations of disease prevalence were recorded between animals of different, age, sex and immune and health status but it was obvious that FMD was more prominent and prevalent in buffaloes (47.1%) than in cattle (34.1%). Therefore, control efforts should focus on reducing the circulation of FMDV among susceptible livestock with special attention towards water buffaloes. Continuous surveillance, at molecular and immunological levels, of FMDV serotypes is needed for the effectiveness of any adopted control strategy targeting FMD including vaccination.