Dirofilaria immitis is an important nematode of dogs and cats which causes fatal heartworm disease in their hosts. The aim of this study was to investigate the prevalence of D. immitis by serologic and molecular methods in shelter dogs, in the Thrace Region of Turkey. Blood samples were collected from 402 dogs in shelters which were located in four cities (Istanbul, Edirne, Tekirdag and Kirklareli) in this region. The blood smears were examined for the presence of D. immitis microfilariae under the light microscope. The commercial Snap3Dx test kit and PCR assay for amplifying the ITS-2 gene region were used for the serological and molecular analyses, respectively. The serologic and molecular prevalence of D. immitis was determined as 6.7% and 2.7%, respectively. D. immitis microfilariae were also determined in the blood smears of three dogs (0.8%). The distribution of the infection according to the provinces was 14.7% in Edirne, 11% in Kirklareli, 1% in Tekirdag and 0% in Istanbul. The occult infection rate was determined as 59.3%. This study revealed the presence of D. immitis for the first time in the Thrace Region of Turkey. This region is the transition point to Europe and obtained data in this study could contribute to the control of heartworm disease in the area.