In Belgium, an official control plan was established in 2016 to detect the potential presence of an unauthorized genetically modified (GM) Bacillus subtilis RASFF2014.1249 strain in commercialized feed additive vitamin B2 products. To this end, two real-time PCR markers specific to this unauthorized genetically modified microorganism (GMM), named UGMVit-B2 and 558, were used. In the present study, the first four-year results from 67 feed additive vitamin B2 samples from the official control are presented. It includes 5 samples positive for real-time PCR methods specific to the unauthorized GM B. subtilis RASFF2014.1249 strain and has led to the RASFF2018.2755 and RASFF2019.3216 notifications. Moreover, a retrospective study using the same feed additive vitamin B2 samples was performed, allowing to provide a first picture of GM bacterial contaminations. It consisted in a first-line screening strategy gathering available PCR-based methods targeting both the B. subtilis species, frequently used to produce vitamin B2, and a set of antimicrobial resistance (AMR) genes commonly harboured as selection marker by GM bacteria used to produce microbial fermentation products. On this basis, suspicious samples contaminated with additional unknown GM bacterial strains as well as potential health and environmental risks related to the unexpected presence of full-length AMR genes could be highlighted. In addition, the possible complementary use of additional data, like chloramphenicol presence and DNA concentration, as indicators for GMM contaminations was assessed. Based on results generated in the present study, the relevance to use the proposed first-line screening strategy supplemented by indicators in order to strengthen the current control strategy was emphasized.