Germany has been free from foot-and-mouth disease virus (FMDV) without vaccination since 1992, but diagnostic capability at regional laboratories is maintained for FMDV exclusion in suspect cases and as surge capacity for outbreak preparedness. A proficiency test was initiated in 2015 to evaluate the diagnostic performance of 20 regional veterinary laboratories. A panel of two identical samples of FMDV genome for real-time reverse transcription polymerase chain reaction (RT-PCR), four lyophilized bovine sera for antibody detection and eight samples of inactivated vaccine antigen for analysis with a lateral-flow device (LFD) were tested with the systems routinely used at the participating institutions. With only one exception, all laboratories reliably detected viral RNA with two real-time RT-PCR assays down to a dilution of 10(-4) of the original material. The LFD pen-side test was evaluated at 8 of 20 laboratories, and FMDV antigen was detected by all participants down to a dilution of 1:81. Serological diagnosis was also very consistent at most participating institutions. Very few false-negative results were returned for the diluted positive sera, and testing of a large cohort of negative samples demonstrated a high specificity of over 99% for the two commercial ELISA kits used (PrioCHECK® FMDV NS and ID Screen® FMD NSP). In conclusion, the proficiency test demonstrated the reliable and robust FMDV diagnostic capability of the German regional veterinary diagnostic laboratories. Furthermore, the suitability of the commercially available ELISA systems for the detection of FMDV-specific antibodies was confirmed. Proficiency tests are an important quality assurance measure for transboundary diseases like FMDV where diagnostic capacity in free countries has to be available at the highest possible standard.