Abstract Serology is used to predict vaccine induced protection against challenge with a heterologous strain of the same serotype of foot-and-mouth disease virus (FMDV). To evaluate the accuracy of such predictions, we compared the protection afforded to cattle vaccinated with the O1 Manisa strain of FMDV against challenge with either a homologous (O1 Manisa) or a heterologous strain (O1 Campos). Serology by virus neutralization test (VNT) using O1 Manisa antiserum predicted an acceptable protection against such a challenge. Two experiments were carried out to compare the results for consistency. A total of 78 naïve cattle were vaccinated with different antigen payloads (60–0.94 μg) of O1 Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O1 Manisa or O1 Campos. Unvaccinated naïve control cattle ( n = 20) were also challenged with either the O1 Manisa or O1 Campos viruses and all developed generalized FMD. The protection results for the vaccinated cattle revealed that higher payloads of O1 Manisa vaccine were needed to protect against heterologous challenge compared to that for homologous challenge. The 50% protective dose (PD50 ) values for the vaccine in experiments 1 and 2 were found to be 28.78 and 9.44 for the homologous challenge and 3.98 and 5.01 for heterologous challenge. Furthermore, protection against O1 Campos required a higher level of vaccine-induced antibody against this virus compared to the level of O1 Manisa neutralizing antibody associated with protection against homologous challenge. The 50% protective level of in vitro neutralizing antibody was found to be log10 1.827 for O1 Campos and log10 0.954 for O1 Manisa based on O1 Manisa based virus neutralization test.