BACKGROUND Rates of multidrug-resistant gram-negative organisms are surpassing those of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci in nursing homes (NHs). OBJECTIVE To characterize the incidence and duration of carriage of ciprofloxacin-resistant Escherichia coli (CipREc) in NHs and identify those in the O25b-ST131 lineage. METHODS We collected 227 CipREc isolates obtained by routine and regular surveillance of high-risk NH residents with indwelling devices. Repetitive element palindromic (REP)-polymerase chain reaction assay and multiplex polymerase chain reaction amplification for O25b-ST131 E. coli detection were performed using (GTG)5-primers and O25pabBspe and trpA2 primer pairs, respectively. RESULTS We found a high period prevalence of CipREc colonization (21.5%), high rates of recolonization with the same strain following clearing (0.46 recolonizations/ person/ year), and an acquisition incidence of 1.05 cases/1,000 person-days. Almost three-quarters of colonized residents carried strains in the O25b-ST131 E. coli lineage. Compared with isolates not in the lineage, O25b-ST131 isolates were carried significantly longer (10 vs 3 months). We identified 18 different REP-types; 2 occurred in 55% of the residents colonized with CipREc, and in more than 1 NH. Duration of CipREc carriage varied by REP-type and averaged 6 months. CONCLUSION CipREc occurred frequently in NH residents and is carried for long durations, and reacquisition following clearance is common Trial registration. ClinicalTrials.gov identifier: NCT01062841.