Analytical methods are needed for the identification and quantification of meat species to detect food adulteration. Since game meat is more expensive than meat from domesticated animal species, it is a potential target for adulteration. We present a tetraplex real-time PCR assay that allows the simultaneous determination of the content of roe deer, red deer, fallow deer and sika deer. The tetraplex assay showed only moderate cross-reactivity with closely related species. After optimization the tetraplex assay had a limit of detection of 0.1% (w/w) and a limit of quantification of 0.5% (w/w) for each of the four deer species. The tetraplex assay was found to be robust, slight modifications of the experimental setup did not lower its performance. Recoveries obtained by analyzing DNA mixtures and DNA isolates from model game sausages were similar to those obtained with the singleplex assays.