The emergence of the plasmid-borne polymyxin resistance gene mcr-1 threatens the clinical utility of last-line polymyxins. Although mcr-1 has disseminated to various Enterobacterales species, the prevalence of mcr-1 is the highest among Escherichia coli isolates while remaining low in Klebsiella pneumoniae. The reason for such a difference in prevalence has not been investigated. In this study, we examined and compared the biological characteristics of various mcr-1 plasmids in these two bacterial species. Although mcr-1-bearing plasmids were stably maintained in both E. coli and K. pneumoniae, the former presented itself to be superior by demonstrating a fitness advantage while carrying the plasmid. The inter- and intraspecies transferability efficiencies were evaluated for common mcr-1-harboring plasmids (IncX4, IncI2, IncHI2, IncP, and IncF types) with native E. coli and K. pneumoniae strains as donors. Here, we found that the conjugation frequencies of mcr-1 plasmids were significantly higher in E. coli than in K. pneumoniae, regardless of the donor species and Inc types of the mcr-1 plasmids. Plasmid invasion experiments revealed that mcr-1 plasmids displayed greater invasiveness and stability in E. coli than in K. pneumoniae. Moreover, K. pneumoniae carrying mcr-1 plasmids showed a competitive disadvantage when cocultured with E. coli. These findings indicate that mcr-1 plasmids could spread more easily among E. coli than among K. pneumoniae isolates and that mcr-1 plasmid-carrying E. coli has a competitive advantage over K. pneumoniae, leading to E. coli being the main mcr-1 reservoir. IMPORTANCE As infections caused by multidrug-resistant "superbugs" are increasing globally, polymyxins are often the only viable therapeutic option. Alarmingly, the wide spread of the plasmid-mediated polymyxin resistance gene mcr-1 is restricting the clinical utility of this last-line treatment option. With this, there is an urgent need to investigate the factors contributing to the spread and persistence of mcr-1-bearing plasmids in the bacterial community. Our research highlights that the higher prevalence of mcr-1 in E. coli than in K. pneumoniae is attributed to the greater transferability and persistence of mcr-1-bearing plasmid in the former species. By gaining these important insights into the persistence of mcr-1 in different bacterial species, we will be able to formulate effective strategies to curb the spread of mcr-1 and prolong the clinical life span of polymyxins.