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 O 1 Manisa strain of FMDV against challenge with either a homologous (O 1 Manisa) or a heterologous strain (O 1 Campos). Serology by virus neutralization test (VNT) using O 1 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 O 1 Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O 1 Manisa or O 1 Campos. Unvaccinated naïve control cattle ( n = 20) were also challenged with either the O 1 Manisa or O 1 Campos viruses and all developed generalized FMD. The protection results for the vaccinated cattle revealed that higher payloads of O 1 Manisa vaccine were needed to protect against heterologous challenge compared to that for homologous challenge. The 50% protective dose (PD 50) 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 O 1 Campos required a higher level of vaccine-induced antibody against this virus compared to the level of O 1 Manisa neutralizing antibody associated with protection against homologous challenge. The 50% protective level of in vitro neutralizing antibody was found to be log 10 1.827 for O 1 Campos and log 10 0.954 for O 1 Manisa based on O 1 Manisa based virus neutralization test.