Polymerase chain reaction (PCR) has been widely used due to its high specificity, sensitivity, and rapid turn-around time. However, inhibitory factors may be co-extracted with the target nucleic acid that will hinder the performance of PCR. In this study, DNA extraction methods for Mycobacterium avium subsp. paratuberculosis were evaluated including rapid lysis, organic extraction, silica-based and magnetic particle-based (MagaZorb) technologies on bacterial cells, and spiked bovine feces. Efficiency of the extraction was determined by PCR end point titration with primers targeting the insertion sequence, IS900. Results of the end point titrations are identical for bacterial cells and spiked feces. Inhibition was observed in PCR with DNA isolated from spiked feces, and a 1/100 dilution was able to alleviate this problem with DNA extracted by MagaZorb. A 1/1000 dilution was required for the other three methods. MagaZorb proved to be more efficient at removing inhibitory factors and required the least labor and completion time. Further evaluation is required for its utilization in other clinical specimens.