A real-time polymerase chain reaction (PCR) using a green fluorescence dye, followed by a comparison of derivative melting curves in the post-amplification phase, was developed to distinguish species of Culicoides within the Obsoletus Complex. The selected target sequence was internal transcribed spacer 2 (ITS 2) of the ribosomal DNA (rDNA). Using the newly developed method, 140 midges were morphologically classified in the Obsoletus Complex and were processed. The results were compared to those obtained by combining the morphological identification with the gel based reverse transcriptase (RT)-PCR. By analysing the species-specific pattern of the dissociation curves, it was possible to identify 52 midges as Culicoides scoticus, 82 midges as C. obsoletus sensu strictu and 6 as C. montanus. These results matched those obtained by the combination of gel-based PCR and morphological identification used on a routine basis. Given its diagnostic flexibility, rapid results, automation capability, high quality result performance and expression, the real-time ITS 2 rDNA PCR appears to be more functional and efficient than the gel-based PCR, especially when dealing with large-scale monitoring of midges belonging to the Obsoletus Complex.