Paratuberculosis is a chronic disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Most economic losses due to MAP occur in the dairy industry. However, the infection is not restricted to cattle, but also occurs in other ruminants, such as sheep, goat, and deer. Although deer are of minimal economic importance in The Netherlands, they may constitute a source of infection for the dairy industry. This pilot study was conducted to estimate the prevalence of Johne's disease in farmed red deer in The Netherlands. Serum and faecal samples were collected from 140 animals, originating from 8 different farms. Four of the farms had animals that tested positive for Johne's disease. The within-herd MAP seroprevalence varied between 4.8% and 21.2%. In conclusion, this pilot study provides evidence of MAP infection in the Dutch farmed deer population, and thus there might be a risk of MAP transmission between farmed red deer and dairy cattle.