Coxiella burnetii (C. burnetii) is an intracellular bacterium and the cause of query fever (Q fever), which is a serious zoonotic disease that influences numerous animal species globally. Thus, the current investigation's aims were to ascertain the molecular diagnosis of C. burnetii and the epidemiological findings' correlation with C. burnetii infection. The present investigation was carried out on 100 dairy cows from the Faculty of Agriculture farm, individual farmer houses from El-Fateh and Abnoub cities, and individual cases investigated in the Veterinary Teaching Hospital in Assiut Governorate, Egypt. Milk samples were collected and examined by California Mastitis Test (CMT), and polymerase chain reaction (PCR) to diagnose C. burnetii. Results showed that 5%, 9%, 11%, and 75% of 100 milk samples were CMT (++), CMT (+), suspicious, and negative, respectively. C. burnetii DNA was reported in the milk of six dairy cows. The percentages of C. burnetii infection had no discernible differences (P<0.05) with locality, age, breed (native and mixed breed), tick infestation, housing system, and health status of molecularly tested dairy cows. Therefore, the frequency of C. burnetii infection in dairy herds emphasizes the critical need for surveillance and adequate biosecurity measures in place to prevent and restrict the spread of Q fever in the Assiut Governorate.