This study describes the molecular detection of human brucellosis among patients with pyrexia of unknown origin. It was a cross-sectional descriptive study and was carried out in the Department of Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh. Non-probability purposive type of sampling technique was used. Blood samples were collected from 400 pyretic patients from September 2018 to August 2019. BCSP31 Brucella genus-specific TaqMan real-time PCR and SYBR Green real-time PCR were undertaken for molecular detection. Out of 400 samples, 22 (5.5%) samples found BCSP31 Brucella genus-specific real-time PCR positive. The study revealed that a considerable number of brucellosis is present in rural areas among risk as well as non-risk group study population having definite male predominancy, most prone to develop among >40-80 years age group. Brucella genus and species-specific real-time PCR might be performed for confirmation and also to avoid unjustified costs, drug toxicity, and un-masking of other potentially dangerous diseases.