Mycobacterium paratuberculosis(MAP) is an aetiology factor in paratuberculosis in ruminants, both domestic and wild. Animals are the reservoir of MAP in the environment and the disease is mainly transmitted to the environment in faeces and milk of sick animals and of those which are infected without symptoms. The presence of mycobacteria in milk may be a source of infection for calves, but also for other animal species and for humans. Mycobacteria are introduced into milk in two ways: with macrophages when animals are infected and by contamination with faeces. In practical terms, faeces contamination is more likely and it increases the MAP count in milk to a greater extent. It has been proven that the HTST (high-temperature short-time) pasteurisation, commonly applied in dairy industry, fails to deactivate mycobacteria completely, which confirms their considerable resistance to high temperatures. The threat related to the mycobacteria presence in milk requires that studies should be conducted in its occurrence. MAP isolation from milk meets with a number of obstacles related to complex three-phase structure of milk on the one hand and the diversity of microorganisms on the other. MAP can be detected by either of two methods: direct isolation of DNA-MAP with the use of QIAamp DNA Mini Kit manufactured by Qiagen or culturing on HEYM substrate. 87 samples of udder milk were examined, taken from milk cows over three years old, belonging to a stock in which cases of subclinical type of paratuberculosis had been found earlier. DNA isolation was conducted according to the manufacturer's recommendations and the obtained solution was subjected to the PCR. MAP genetic material was found in 21 (24.1%) of the udder milk samples. When isolating MAP in the culturing method on the HEYM substrate, having first decontaminated and made milk samples uniform, 43 strains were cultured with the morphological features typical of genus Mycobacterium; however, the presence of an insertion fragment IS-900 was confirmed only in two cases. 18 samples of milk gave a positive result of direct isolation of DNA-MAP and an increase in the number of colonies typical of genus Mycobacterium on the HEYM substrate; in one case, no growth was observed despite the presence of DNA-MAP. Analysing udder milk for the DNA-MAP presence may be used as complement of diagnostic tests.