Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne`s disease (JD) in ruminants. This is the first large scale report to estimate the herd-level prevalence of antibodies against MAP by using an ELISA to detect antibodies in bulk tank milk (BTM) samples. The samples were collected from January 2011 to November 2011, from 636 herds of the dairy farms in the Gyeonggi and Chungbuk areas of Korea. The overall apparent prevalence of MAP antibody-positive herds was 8.5%, and regional prevalence were 32/440 (7.3%) and 22/196 (11.2%) of dairy farms in the Gyeonggi and Chungbuk areas, respectively. The results did not differ significantly by region. While we have determined the prevalence rate of MAP in the Gyenoggi and Chungbuk areas in this study, there is a continuing need for well-designed studies to calculate the prevalence of MAP in dairy herds based on culture and molecular findings.