It is important to develop the polymerase chain reaction (P.C.R.) and the restriction fragments of longitude polymorphism (R.F.L.P.) technologies to identify genome segments since they constitute quick, sensitive, and specific methodologies for the detection of Mycobacterium avium subsp. paratuberculosis, a microorganism that produces animal Paratuberculosis and that is related to Crohn's disease in humans. Objective: isolate M. paratuberculosis strains and to apply a P.C.R. protocol to confirm the IS900 insertion sequence, by R.F.L.P the genetic pattern of the strains isolated and to analyze cellular and extracellular proteins expressed in M. paratuberculosis. Twelve M. paratuberculosis strains, the strains isolated developed only in mycobactin Herrold medium, showing their characteristic dependence. The analysis by P.C.R. was positive starting from strains developed in the culture medium. When faeces were inoculated with a M. paratuberculosis strain, the detection rate was of 100%, demonstrating the highest specificity for IS900 in a 1:1000 dilution of the problem sample, but it was negative starting from the raw sample (faeces). All the isolates revealed an identical R.F.L.P. pattern, with a 217-bp probe located to the right of the cut of the BstE11 endonuclease. All of the isolates analyzed from this deer farm possessed R.F.L.P.-BstE11 "A" pattern. Inmmunoblotting detected 65 kDa (thermal shock), 42 kDa, 35 kDa, and 28 kDa protein antigens, from the bacterial extracts as well as directly from the tissues corresponding to such an isolate from an animal with clinical symptoms and characteristic lesions of Paratuberculosis in the organs. The negative results of the immunoblotting of most of the strains cultured could be because the mycobacterial growth was not enough to detect the proteins. However, these tests could be the basis of a program to control Paratuberculosis in deer, especially if multiple tests are used to ensure that as many as possible of the infected animals are detected.