Technical Abstract: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a chronic enteric infection that affects ruminants. Despite the ubiquitous occurrence of Mycobacterium sp. in nature and the fact that Johne’s disease has been reported worldwide, little research has been done to assess its survival in agricultural environments. The goal of this 365 day study was to evaluate the ability of M. paratuberculosis to persist in mixed-community biofilms on coupons composed of common livestock watering trough materials. M. paratuberculosis was inoculated into triplicate containers with 32L of trough water and either concrete, plastic, galvanized or stainless steel coupons. The concentration of M. paratuberculosis was determined by using quantitative, real-time PCR to target the IS900 sequence in DNA extracts. High concentrations of M. paratuberculosis were detected within the biofilms after three days (around 1 X 105 cells per cm2). Based on the best-fit slopes, the time required for a 99% reduction (t99) in coupon-associated M. paratuberculosis cells were 1.1 x 106, 7.5 x 104, 46 and 27 days for galvanized steel, concrete, plastic and stainless steel trough materials, respectively. In general, M. paratuberculosis concentrations on stainless steel and plastic trough materials were one to two orders of magnitude lower than the concentration on the other trough materials. These results suggest that M. paratuberculosis survives well in biofilms present on trough materials. Trough material composition influenced the survival of M. paratuberculosis with the lowest survival exhibited on stainless steel, followed by plastic, galvanized steel and concrete.