In this research, we investigate the prevalence of Dirofilaria immitis infection in dogs using microscopical, serological, and molecular methods from the Central Anatolia Region (Sivas) of Turkey and reveal the associated risk factors. Whole blood and serum samples were obtained from 306 dogs older than 7 months. Polymerase chain reaction (PCR) and the modified Knott's method (Knott) were used for detecting the microfilaria and enzyme-linked immunosorbent assay (ELISA) was applied for the presence of D. immitis antigens. The prevalence of D. immitis in dogs was detected as 2.9% (9/306) by PCR and ELISA and as 1.3% (4/306) by Knott. All Knottpositive samples were also found positive by both PCR and ELISA. According to ELISA and PCR results, no statistically significant difference was found between the prevalence in males (4.2%) and females (1.5%); dogs 0.7-2 years old (2.5%), 3-5 years old (2.2%); and more than 6 years old (7.1%); or stray (3.7%) and owned (1.7%) dogs (P > 0.05). One of the PCR products was selected for sequence analyses of the 16S small subunit rRNA gene and the obtained sequence was submitted to GenBank (KJ183078). The 16S rRNA sequence exhibited 100% identity to other D. immitis sequences available in GenBank (FJ799911, FJ799917, FJ799916). This research provides the first data on D. immitis infections in dogs from Sivas Province by using the combined identification techniques.