Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis (PTB). The first step in the control of PTB is the identification and isolation of sub-clinical fecal shedders from the herd. In the current study, real-time and nested PCR targeting MAP-specific genetic elements (IS900 and ISMAP02) DNA isolated from fecal samples were used to detect MAP infection in cattle. Of the 1,562 fecal samples obtained from 37 herds, regardless of diarrhea, 35 samples tested positive in both IS900-targeted real-time and ISMAP02-targeted nested PCR. At the herd level, 12 of the 37 herds were found to be positive for MAP. Detection rates of the PCR tests were similar to those reported for ELISA-based methods. These results suggest that PCR can be used to detect sub-clinical fecal shedders of MAP.