Aims: To compare the two different diagnostic assays for the detection of Mycobacterium avium ssp. paratuberculosis, the aetiological agent of paratuberculosis. Methods and Results: Faecal samples were derived from 310 cows, representing 13 commercial dairy herds in various locations in Switzerland with expected increased risk because of a past history of disease. Detection assays for M. avium ssp. paratuberculosis were culture (gold standard) and a newly designed real-time PCR. Real-time PCR identified 31 of 310 animals as positive within this risk population whereas culture identified 20 positive animals. The specificity of real-time PCR was confirmed by DNA sequencing of the PCR product. Depending on the test used, the paratuberculosis prevalence in our tested risk population ranged from 6.5 to 10%. Conclusions: Real-time PCR and culture data were in good agreement, and real-time PCR generates data in a short time in contrast to culture. Significance and Impact of the Study: We consider real-time PCR as a suitable alternative method to culture for the detection of M. avium ssp. paratuberculosis in a national surveillance programme.