Mycobacterium avium ssp. paratuberculosis (MAP) causes ruminant paratuberculosis (Johne's disease) worldwide. Oral-fecal contamination is the most important mode of transmission of paratuberculosis, so eradicating MAP-shedding animals could prevent disease propagation. Fecal culture, a well-known method for MAP diagnosis, requires costly specialized media and a long incubation time that sometimes ends in disappointing bacterial contamination. To facilitate the efforts of control programs, we evaluated the performance of direct fecal quantitative PCR (qPCR) assays for their sensitivity and robustness for MAP detection. Commercial kits use different strategies for extracting DNA, combined with qPCR systems, to detect the presence of MAP in fecal samples. In this study, we compared the sensitivity of 3 commercially available DNA extraction kits (A, B, and C) combined with 2 qPCR systems (T and V) for the detection of MAP in infectious cows. A total of 49 dairy cows from 5 herds were sampled twice a year for 3 yr and diagnosed using fecal culture and ELISA. Eight replicates of their fecal samples from the first sampling were tested using each DNA extraction method and qPCR detection system. Although all 3 of the commercial DNA extraction kits have been previously described as very efficient for the diagnosis of paratuberculosis, kit B provided the highest sensitivity. Indeed, 89% of the cows declared positive for paratuberculosis by both fecal culture and ELISA were identified with kit B, whereas only 23 and 43% of the cows were identified with kits A and C, respectively. Interestingly, kit B was able to detect some low-MAP shedders. The qPCR detection system also played a critical role: system T yielded qPCR with the highest sensitivity. The results of this study suggest that DNA extraction kit B combined with detection system T provides the best amplification of MAP DNA from fecal samples with the highest sensitivity and specificity. Although 1 DNA extraction and qPCR analysis should be adequate to confirm that an animal with diarrhea or other signs of paratuberculosis is positive, detecting low shedders at the highest sensitivity should include repetitive testing. This study demonstrates the importance of repetitions using the most appropriate method for extracting DNA from fecal samples, combined with a compatible qPCR system for identifying MAP-shedding animals.