Mycobacterium avium ssp. paratuberculosis (MAP) is the causative organism of Johne's disease. Although fecal culture is considered the standard diagnostic test, the long incubation times, costs, and intermittent shedding of MAP hinder efficient screening programs based on culture results. The primary objectives of this study were to determine the detection ability of solid culture, broth culture, and real-time PCR (qPCR) for MAP in fecal samples and to assess how shedding patterns of MAP in feces vary with lactation stage and season. This knowledge could improve the use of these diagnostic assays in Johne's management programs. For this study, 51 MAP-infectious cows from 7 Atlantic Canadian dairy farms had fecal samples collected monthly over a 12-mo period. Samples were analyzed for MAP bacterial load via solid culture, broth culture, and qPCR. For all fecal samples, 46% [95% confidence interval (CI): 40 to 51%] were positive by solid culture, 55% (95% CI: 50 to 60%) by broth culture, and 78% (95% CI: 73 to 82%) by qPCR. Sensitivity of qPCR was numerically higher in the dry and postpartum lactation periods, and qPCR detection in summer and fall was 85% of that in winter and spring. Furthermore, culture-determined moderate or light shedding categories generally corresponded to qPCR cycle threshold values <35, but heavy shedding categories corresponded to qPCR values <29. Direct fecal qPCR is a MAP detection method that is quick and less costly than culture techniques, and it avoids the use of decontamination steps that could decrease numbers of bacteria in a sample below the detection limit. This study indicates that, for known MAP-positive cows, fecal qPCR had high sensitivity of MAP detection, thereby supporting the use of direct fecal qPCR as part of a Johne's herd control program.