Chronic wasting disease (CWD) is a highly contagious prion disease occurring in free-ranging and farmed cervids. CWD continues to spread uncontrolled across North America, and cases continue to be detected almost every year in the Republic of Korea. CWD-infected animals contaminate the soil by releasing infectious prions through their excreta, and shed prions accumulate and remain infectious in the soil for years. Given that the upper soil levels can become contaminated with prions and serve as infectivity reservoirs facilitating horizontal transmission of CWD, the ability to detect prions in the soil is needed for monitoring and managing CWD spread. Using the protein misfolding cyclic amplification (PMCA) technique, we investigated whether prions could be amplified and detected in farm soil experimentally exposed to CWD-infected brain homogenate as well as in the soil of CWD-affected farms. From each soil sample, we performed 10 serial extractions and used these 10 extracts as PMCA templates. Here, we show that prion seeding activity was detected in extracts from farm soil following 4 years of incubation with CWD-infected brain homogenate. More importantly, 13 of 38 soil samples collected from six CWD-affected farms displayed prion seeding activity, with at least one soil sample in each farm being PMCA positive. Mouse bioassays confirmed the presence of prion infectivity in the soil extracts in which PMCA seeding activity was detected. This is the first report describing the successful detection of prions in soil collected from CWD-affected farms, suggesting that PMCA conducted on serial soil extracts is a sensitive means for prion detection in CWD-contaminated soil.IMPORTANCEChronic wasting disease (CWD) is a highly contagious prion disease affecting free-ranging and farmed cervids. CWD continues to spread uncontrollably across North America, and multiple cases are detected annually in the Republic of Korea. Prions shed from CWD-infected animals remain infectious in the soil for years, serving as infectivity reservoirs that facilitate horizontal transmission of the disease. Therefore, the ability to detect CWD prions in soil is crucial for monitoring and managing the spread of the disease. In this study, we have demonstrated for the first time that prions in the soil of CWD-affected farms can be reliably detected using a combination of serial soil extraction and a prion amplification technique. Our data, in which at least one soil sample tested positive for CWD in each of the six CWD-affected farms analyzed, suggest that the approach employed in this study is a sensitive method for prion detection in CWD-contaminated soil.