A serologic study was conducted to determine the seroprevalence of Mycobacterium paratuberculosis in dairy herds and cows, in the most important dairy region of Uruguay. At 1945, Casamagnani isolated the agent and made the first report of clinical disease in Uruguay. At 1983, Errico made the last report of this disease, in the country. The objective of this study was to estimate the serologic prevalence and the spread of the disease among herds. The dairy population in the study region is 2000 herds with 180000 milking cows. A two step random sample was drawn from the population. In the first step 36 dairy herds were selected and in the second step 20 milking cows were selected in each farm by systematic sampling. The total number of studied cows was 720. Data were evaluated in the analysis weighted by the number of milking cows sampled using the routine of Intercooled Stata 7.0. Sera were tested with an ELISA kit from IDEXX Lab Inc. Apparent prevalence was 16.02% ( 2.58. A positive herd was defined as any herd with two or more ELISA positive cows. The proportion of positive herds was 72% (26/36). In conclusion, the disease is present in Uruguay with a high spread among herds. Therefore, it is recommend to start developing control programs.