PCR technique was performed on faecal material and tissue samples taken post-mortem from the same animals in order to make paratuberculosis diagnosis in different ruminant species. The samples were taken from an herd in a countryside around Rome. We investigated serologically positive cattle, goats and water-buffaloes with specific symptoms of Johne's disease. Other animals with not specific signs of the pathology were analysed. Biomolecular tests were compared with classical serological and microbiological methods. For PCR test, specific oligonucleotide primers were chosen inner to IS 900 sequence to amplify a 400-bp fragment. To confirm the results a 229-bp was designated by nested PCR from the first amplification product. Sensitivity and specificity of the methods were established. The infection can spread in environment and pass through different animal species living in the same farm. Biomolecular technique may be utilised as tool for validation of paratuberculosis diagnosis with different methods and for detection of latent forms of disease. It may be useful to avoid the spreading of infection in the farms and in the environment.