This Technical Specification describes procedures for DNA extraction from alfalfa seeds and for the event-specific detection of the alfalfa lines J101, J163 and KK179. The detection methods are based on real-time PCR and are targeting the DNA transition sequences between the alfalfa genome and the respective integrated gene construct. The methods can be applied for direct event-specific identification or as a follow-up analysis, if sequences encoding the promoter of the figwort mosaic virus (P-FMV), the terminator of the nopaline synthase gene from Rhizobium radiobacter (T-nos), or the construct CTP2-CP4-EPSPS (herbicide tolerance) were detected by screening analyses of alfalfa samples. In this part of ISO 21569 the DNA extraction method was validated using ground alfalfa seeds and the PCR methods using DNA extracted from seeds. The PCR methods may also be used for the analysis of other matrices (e.g. feed- and foodstuffs). The application of the PCR methods require the extraction of an adequate amount of amplifiable DNA from the relevant matrix.