Antimicrobial resistance is a global threat to public health, requiring a multisectoral "One Health" approach to mitigate this “silent pandemic”. This has been aggravated due to the inappropriate use of antibiotics. Food-producing animals are one of the main contributors to antibiotic usage annually. The dissemination of antibiotic-resistant bacteria, such as Enterobacteriaceae, can occur from animals, acting as reservoirs, to the environment through contaminated feces, or to humans through the food chain. These bacteria are considered priority pathogens worldwide based on carrying antimicrobial-resistant genes. Pioneering results in Portuguese native cattle breeds demonstrated a phenotypic profile of multidrug-resistant E. coli. To better understand the role of native breeds as reservoirs of antibiotic-resistant bacteria, the main objective of the EnteroGEN project is to characterize the genetic resistance mechanisms in Enterobacteriaceae isolated from cattle of different breeds like Holstein-Friesian and, the iconic Portuguese native breeds such as Barrosã, Cachena and Minhota.