Abstract: The main objective of the present research was to investigate rate of contamination to MAP in a dairy herd with long history of contamination. Two hundred and thirteen dairy cows were randomly selected from a herd in Mashhad, Iran and were tested for the presence of MAP. After determination of the presence of MAP from fecal samples by PCR technique, reproductive and production indices of the positive and negative cases were compared. Fecal samples were collected via rectum of the selected cows and DNA was extracted from feces with Bioneer Kit (South Korea). According to the results from MAP genome project, specific element, namely, ISMAP02 that had 6 copies in the bacterial genome was detected. In order to increase sensitivity of the test, Nested-PCR was used, therefore, a pair of primer was used for the first PCR and another pair was used for Nested-PCR. Specificity of these primers allowed us to detect MAP only, while other primers including IS900 don’t have the ability to differentiate some of the non pathogenic species. Out of 213 cows, 25 cases tested positive. Cows were divided into two groups of ≤2 and ≥3 parities. Production and reproduction factors were analyzed statistically by SPSS package. Kolmogrove-Smirnov and Shapiro-Wilk tests determined that sample population was not normal, therefore, Mann-Whitney test was used for the statistical analysis of all the factors. For the comparison of fetal absorption, abortion and stillbirth, χ²-test was used. Because of the low numbers of culled cows (below 5% of the total) in the positive cases in one of the groups, Fisher’s exact test was used. Results showed that in 112 cows of the first group, 13 cows and in the 101 cows of the second group 12 cows were tested positive. Milk production in the last milking period in the positive cows of the group 1 was less than the negative cows (p<0.05). Rate of stillbirth and abortion in both groups were higher in infected cows than in non infected group (p<0.05). DO and CI was higher in infected than non infected cows but, the difference was significant in DO of the second group only (p<0.05). Other reproductive factors were not different (p>0.05), but, in general, infected cows had lower reproductive performance as compared to the non infected cows. A highly significant difference was observed in the culling rate of the infected cows of both groups (p<0.000 for the 1st group) and (p<0.02 for the 2nd group) as compared with culling rate of the non infected cows. With regard to the similarity of contamination rate in both groups, it can be inferred that contamination to MAP was widely spread in the herd. Moreover, infected cows had lower reproductive performance than non-infected cows.