This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10-7. Based on the peak CT values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100-1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance.