The outbreak of bluetongue disease in Central Europe necessitates new approaches in the identification of vectors to follow-up changes of populations of species and not of complexes. Since females of species of the complex of Culicoides obsoletus are difficult to be identified according to morphological criteria, we applied a polymerase chain reaction (PCR)-based strategy targeting the mitochondrial cytochrome oxidase subunit I to differentiate between the species Culicoides obsoletus s.s. and Culicoides scoticus. Catches of culicoids obtained from May to November 2007 in an ultraviolet lamp trap at a cattle farm in Rhineland-Palatinate, Southern Germany were surveyed for changes of the abundance of both species. Only in May 2007, the samples contained similar proportions of both species. Afterwards, C. scoticus dominated with up to 88%. Calculating the number of specimens of both species within the total catches of culicoids, the numbers of C. obsoletus s.s. slightly decreased from May to July and increased to a little maximum in August. C. scoticus seemed to have three maxima in this period of time, the strongest one in August, presumably due to different generations and not to climatic conditions. These results indicate that the applied PCR strategy can be used for a detailed analysis of culicoids as basis for the estimation of the transmission risk of the bluetongue virus by different species of the Obsoletus complex.