AbstractBackgroundReal‐time PCR is the diagnostic technique of choice for the diagnosis and control of equine herpesvirus‐1 (EHV‐1) in an outbreak setting. The presence of EHV‐1 in nasal swabs (NS), whole blood, brain and spinal cord samples has been extensively described; however, there are no reports on the excretion of EHV‐1 in urine, its DNA detection patterns, and the role of urine in viral spread during an outbreak.ObjectivesTo determine the presence of EHV‐1 DNA in urine during natural infection and to compare the DNA detection patterns of EHV‐1 in urine, buffy coat (BC) and NS.Study designDescriptive study of natural infection.MethodsUrine and whole blood/NS samples were collected at different time points during the hospitalisation of 21 horses involved in two EHV‐1 myeloencephalopathy outbreaks in 2021 and 2023 in Spain. Quantitative real‐time PCR was performed to compare the viral DNA load between BC‐urine samples in 2021 and NS‐urine samples in 2023. Sex, age, breed, presence of neurological signs, EHV‐1 vaccination status and treatment data were recorded for all horses.ResultsA total of 18 hospitalised horses during the 2021 and 2023 outbreaks were positive for EHV‐1, and viral DNA was detected in urine samples from a total of 11 horses in both outbreaks. Compared with BC samples, DNA presence was detected in urine samples for longer duration and with slightly higher concentration; however, compared with NS, detection of EHV‐1 in urine was similar in duration with lower DNA concentrations.Main limitationsLimited sample size, different sampling times and protocols (BC vs. NS) in two natural infection outbreak settings.ConclusionsEHV‐1 was detected in the urine from naturally infected horses. Urine should be considered as complimentary to blood and NS in diagnosis of EHV‐1 infection.