The majority of edible gelatin in Europe is derived from pigskin, but a significant portion is extracted from bovine tissue. Because of the bovine spongiform encephalopathy crisis, consumers might be concerned about the gelatin used in various products. To assure consumers of the quality and safety of edible gelatin, European Union directive 1999/724/EC described general guidelines for gelatin production, including requirements for documentary proof confirming that raw materials are from animals fit for human consumption. Analytical methods to confirm gelatin documentation or raw material animal species source in the finished product are lacking. In this study, several published species-specific PCR systems were evaluated as potential molecular methods for determining the origin of the raw material used in making gelatin. A recently validated bovine species-specific PCR primer set targeting the ATPase 8 subunit gene in bovine mitochondrial DNA was suitable for detection of bovine material in gelatin. This PCR primer set was optimized using conventional and real-time PCR approaches. An evaluation of these two PCR methods confirmed the high specificity for the adopted primer set in various gelatin matrices of known origin. The inclusion of bovine gelatin in pork or fish gelatin can be detected at 0.1 to 0.001%. These PCR assays are potential molecular detection tools that can be used to routinely detect bovine gelatin either alone or as an inclusion in gelatin made from other species.