Brucellosis is the most common zoonotic disease in the Mediterranean region where Turkey is located. Therefore, particularly in this region, it is crucial to follow molecular surveillance methods for the control and eradication of this disease agent, which threatens both human and animal life. We have conducted a molecular-based surveillance study for detecting the origin of the possible reservoir in human brucellosis and transmission of Brucella spp. between humans and ruminants. For the detection of clonal relationships, we isolated Brucella spp. from human and ruminants diagnosed with brucellosis and evaluated them by PFGE and MLVA methods. We found 10 subclusters in PFGE and 22 subclusters in MLVA-16. Human and ruminant isolates were found to co-exist in MLVA-16-m1 and PFGE-a8, a9, a10 subcluster by both methods. In conclusion, the results revealed that these subcluster isolates that co-exist some ruminant and human isolates can be more effective in infecting humans.