Serum samples were collected from domestic horses in 4 different regions of Costa Rica to detect antibodies against vesicular stomatitis viruses, serotypes New Jersey (VSV-NJ) and Indiana (VSV-IN). A total of 214 samples were tested by the virus neutralization test. The sampling regions were identified as low North Pacific dry area (1), low Middle Atlantic humid area (2), low South Pacific humid area (3), and the highlands (4). In region 1, 97.1% of horses were positive for VSV-NJ and 16.5% were positive for VSV-IN. The mean antibody titer and its standard deviation after logarithmic transformation were 5.86 ± 0.9 for VSV-NJ and 3.55 ± 1.66 for VSV-IN for region 1. In region 2, 40.1% of horses were positive for VSV-NJ and 32.2% were positive for VSV-IN. The mean antibody titer in region 2 was 4.33 ± 1.82 for VSV-NJ and 3.47 ± 1.73 for VSV-IN. In region 3, 20.79% of horses were positive for VSV-NJ and 27.6% were positive for VSV-IN. The mean antibody titer in region 3 was 4.39 ± 1.89 for VSV-NJ and 3.47 ± 1.82 for VSV-IN. In region 4, 91.3% of horses were positive for VSV-NJ and 73.9% were positive for VSV-IN. The mean antibody titer in region 4 was 5.77 ± 1.10 for VSV-NJ and 4.85 ± 1.63 for VSV-IN. This is the first published report of the detection of virus-neutralizing antibodies against VSV-NJ and VSV-IN in horses in Costa Rica.