A total of 200 Egyptian buffalo calves aging between 9-11 months previously vaccinated with local trivalent oil inactivated vaccine (O, A and SAT-2), in different locations at Menufyia Governorate showing typical clinical signs of FMD were examined for molecular characterization of FMD virus, electrocardiographic picture (ECG) and cardiac biomarkers. The results of serological tests revealed that incidence of non-structural proteins against natural infection with FMDV was 31.5% in serum samples collected from vaccinated buffalo calves. Moreover, 51.5 % of the samples showed detection of structural proteins against trivalent vaccination. On the other hand, there were no protective antibodies against FMDV in 17 % of the examined samples. ECG of FMD infected calves showed ventricular premature depolarization with flattening of P- wave and increased QRS duration with decrease in its amplitude compared with healthy calves. In FMD infected buffalo calves, there were significant (P < 0.01) increases in (cTnI), (CK-MB), urea, CL and K and there was significant (P < 0.01) decrease in Na levels than that of healthy ones. The identified strains in FMD infected Egyptian buffalo calves were unique and different from the vaccinal and other Egyptian strains as well as they were clustered with Topotype ME-SA. Cardiac enzymes along with ECG can be used as useful prognostic tools in FMD infected Egyptian buffalo calves.