AbstractThe emergence ofmobilecolistinresistance (mcr) threatens to undermine the clinical efficacy of the last antibiotic that can be used to treat serious infections caused by Gram-negative pathogens. Here we measure the fitness cost of a newly discovered MCR-3 using in vitro growth and competition assays.mcr-3expression confers a lower fitness cost thanmcr-1, as determined by competitive ability and cell viability. Consistent with these findings, plasmids carryingmcr-3have higher stability thanmcr-1plasmids across a range ofEscherichia colistrains. Crucially,mcr-3plasmids can stably persist, even in the absence of colistin. Recent compensatory evolution has helped to offset the cost ofmcr-3expression, as demonstrated by the high fitness ofmcr-3.5as opposed tomcr-3.1. Reconstructing all of the possible evolutionary trajectories frommcr-3.1tomcr-3.5reveals a complex fitness landscape shaped by negative epistasis between compensatory and neutral mutations. Our findings highlight the importance of fitness costs and compensatory evolution in driving the dynamics and stability of mobile colistin resistance in bacterial populations, and they highlight the need to understand how processes (other than colistin use) impactmcrdynamics.