It is thought that antimicrobial resistance imposes a fitness cost on bacteria, so that a reduction in antimicrobial use may reduce the incidence of resistant bacteria. The objectives of the present study were to determine (1) whether multidrug resistant (MDR) Escherichia coli field strains with different plasmid profiles show disparate plasmid loss when grown over time without selection pressure; (2) whether the number of plasmids present in the cell affects growth. Nine β-hemolytic E. coli strains from swine (n = 8) and cattle (n = 1) were grown in separate continuous-flow vessels for 36 days without antimicrobial selection. Populations were enumerated on brain heart infusion agar and brain heart infusion agar with tetracycline on days 2, 5, 8, 15, 22, 29, and 36. Growth rates, plasmid profiles and susceptibility profiles of the strains were compared, and day 36 isolates (n = 40, five for each MDR strain) were compared with their corresponding day 0 strains. Plasmid content of the nine field strains ranged from zero to eight with sizes from 3.2 to 165 kb. Changes in susceptibility profiles of day 36 isolates were observed among 20% (8 of 40) of the isolates. MDR E. coli largely maintained their original plasmid profiles, replicon types, and susceptibility profiles over 36 days of continuous culture. There was no significant difference in maximum specific growth rate among strains when compared with the plasmid-free strain or when day 36 isolates were compared with their own day 0 strain. This suggests that there is little fitness cost in the maintenance of multiple plasmids of various sizes under the conditions of this study. Other strategies rather than merely reducing antimicrobial usage are needed to combat the emergence of MDR bacteria.