Carbapenem-resistant Enterobacterales (CRE) has been designated as an urgent antibiotic resistance threat by both the U.S. Centers for Disease Control and Prevention and the World Health Organization. The basis for this notoriety is the propensity for CRE to cause outbreaks among vulnerable hospitalized patients, high mortality rates for infected patients, and the emergence of strains of CRE that are resistant to virtually all available antibiotics. The most problematic strains of CRE are those that have become carbapenem-resistant via the acquisition of a carbapenemase gene that can cleave most beta-lactam antibiotics. These carbapenemase genes are most often carried on plasmids, and through conjugation have disseminated widely among diverse members of the Enterobacterales order. Given the great diversity of CRE observed in healthcare settings, a critical need from a public health surveillance perspective is the ability to assess the risk posed by a newly detected strain. We reason that the risk of a newly emerged CRE strain is dictated by: i) the stability of its association with the carbapenemase containing plasmid, ii) it’s capacity to disseminate this plasmid to other Enterobacterales, iii) the potential of the CRE strain to evolve resistance to additional front-line antibiotics, and iv) its potential to colonize, infect and spread among vulnerable patient populations. In this project we experimentally quantify the variation in these four determinants of risk in longitudinal collections of CRE collected from two healthcare networks in the U.S. and employ bacterial genome-wide association studies (bGWAS) to identify genetic variation associated with each of these four phenotypes. We plan to take advantage of the longitudinal nature our sample collections, and the availability of relevant patient meta-data, to quantify the degree to which variation in risk potential predicts patterns of CRE strain emergence, resistance evolution and spread. We expect this project to contribute substantially to our understanding of how genetic variation in CRE influences its epidemic potential.