The aim of this study was to assess the vaccine-matching and antigenic properties of foot-and-mouth disease virus (FMDV) isolates collected from Ethiopia between 2011 and 2014. Samples (n = 51) were collected from cattle and pigs with clinical signs consistent with foot-and-mouth disease (FMD) on farms in Debre-Berhan, Debre-Zeit/Bishoftu, Sidamo, Mekelle, and Addis Ababa. Infectious FMDV was isolated using BHK-21 cell cultures from 38 of the 51 field samples (74.5%). All of these FMDV-positive samples were characterized as serotype O, belonging to two East Africa topotypes (EA-3 and EA-4), and their VP1-encoding sequences demonstrated amino acid sequence variability encompassing 27 positions in comparison to the vaccine strain (O/ETH/38/2005) currently provided by the National Veterinary Institute of Ethiopia. One-dimensional virus neutralization test (1 dm VNT) results showed that O/ETH/38/2005 was antigenically matched to 10 of the 16 serotype O viruses. These findings indicate that the O/ETH/38/2005 vaccine strain can provide protection against outbreaks caused by the O/EA-3 topotype, although poorer vaccine-matching results for the O/EA-4 topotype reinforce the importance of using a good-quality vaccine with high coverage in the susceptible herds with supporting post-vaccination serosurveillance to ensure that sufficient antibody titers are generated in the vaccinated animals.