In this study, the presence of paratuberculosis was investigated by serological and molecular methods in herds of dairy cattle. Blood, milk, and stool samples of 147 cows aged 2 years old or older with chronic diarrhea were collected. A California mastitis test (CMT) was performed on milk samples. Indirect paratuberculosis enzyme-linked immunosorbent assay (ELISA) test was used for serological investigation. Polymerase chain reaction (PCR) was utilized for molecular identification of Mycobacterium avium subsp. paratuberculosis (MAP) from milk and stool samples. Immunomagnetic separation (IMS) method was used for milk and fecal samples. Eighteen (12.24%), 44 (29.93%), 36 (24.49%), 28 (19.05%), and 21 (14.29%) of 147 milk samples were negative, suspicious, CMT (+), CMT (++), and CMT (+++) by CMT, respectively. According to ELISA results, 18 (12.24%) serum samples were positive. In PCR of milk and stool samples, MAP DNA was detected in 20 (13.61%) and 42 (28.57%) of samples, respectively. In IMS PCR assays of the same samples, positivity was not detected. In this study, paratuberculosis was found at high rates in Kayseri. In conclusion, it was detected that mastitis symptoms in paratuberculosis were subclinical and not always observed, and the use of diagnostic laboratory methods may be an important aid in revealing diseases.