Soil is a potential reservoir of human pathogens and a possible source of contamination of animals, crops and water. In order to study the distribution of Listeria monocytogenes in French soils, a real-time PCR TaqMan assay targeting the phosphoribosylpyrophosphate synthetase (prs) gene of L. monocytogenes was developed for the specific detection and quantification of this bacterium within a collection of 1315 soil DNAs originated from the French Soil Quality Monitoring Network. The prs real-time PCR TaqMan assay was specific for L. monocytogenes and could quantify accurately down to 104L. monocytogenes per gram of dry soil. Among the 1315 soil DNAs, prs was not detected. This suggested that the level of L. monocytogenes in French soils is generally less than 104L. monocytogenes per gram of dry soil. In order to confirm this hypothesis, we investigated the occurrence of L. monocytogenes in samples collected in the Burgundy region by culture-based and molecular detection methods on the same samples. By using cultivation-based detection, 17% of samples were positive for the presence of L. monocytogenes while only 2% were found positive by the molecular detection method. L. monocytogenes was repeatedly isolated from cow pasture soils but not from cultivated soils, meadows or forest soils. Isolates were grouped in the serovar 1/2a or 3a and 4b or 4d or 4e. Taken as a whole, molecular detection results globally demonstrate that the level of L. monocytogenes in French soils does not exceed 104 CFU per gram of dry soil. However, in comparison with culture-based method, PCR-based detection underestimates the occurrence of L. monocytogenes in soils. Soil sampling procedure also appears critical and may also lead to the underestimation of the incidence of L. monocytogenes.