Polymerase chain reaction (PCR) has become the main method for detection and identification of genetically modified organisms (GMOs). Multiplex PCR is a variation of this technique allowing simultaneous amplification of several targets in the same reactions. In this study, it was developed, optimized and performed in-house validation of the multiplex PCR method for detection and identification of genetically modified rape lines (GT73, Ms8, Rf3 and T45) in two types of assays: gene- and event-specific. The optimized reactions exhibited high specificity, sensitivity, selectivity and accuracy. The limit of detection (LOD) for the gene-specific reaction was 0.01% for each of the tested lines of rape. For the event-specific reaction, LOD was at the same level for most of the GM rape lines except for T45 (LOD = 0.025%). The optimized and validated assays were employed from 2012 to 2015 in our laboratory to analyse 428 samples of animal feedstuffs containing oilseed rape. GM rape was not detected in the analysed feedstuffs until 2013 (only some traces of GM soyabean were found). Since 2014, the GM rape presence has been confirmed in event-specific reactions in more than one third of analysed samples.