Objective: To analyze the distribution and source of multidrug-resistant organisms (MDRO) infection in non-ICU departments, and provide basis for accurate prevention and control measures of MDRO. Methods: A total of 1116 MDRO infection strains isolated from 802 patients with MDRO infection in the non-ICU departments of The First Affiliated Hospital with Nanjing Medical Universityl from October 2017 to September 2019 were selected as subjects. According to the source, MDRO was divided into two types and four groups: out-of-hospital infection (transferred from outside hospital and community-acquired) and nosocomial infection (transferred from our hospital and department-acquired). Results: The major MDRO infections in non-ICU departments were CRE (34.41%) and MRSA (29.66%). There were significant differences in the composition of MDRO infection among different departments (χ2 = 185.687, P < 0.001). The departments with the most MDRO infection were geriatric medicine (13.17%), neurosurgery (10.04%), and rehabilitation medicine (8.51%). The detection rates of CRE, MRSA, CRAB, and CRPA were 10.69%, 43.83%, 33.72%, and 27.11%, respectively, which were significantly lower than those of CRE, MRSA, CRAB, and CRPA in the departments of the whole hospital (25.35%, 51.48%, 79.15%, 46.99%), and the differences were statistically significant (χ2 = 584.309, 15.583, 960.632, 203.726; all P < 0.001). There were significant differences in the detection rates of four kinds of MDRO among different non-ICU departments (χ2 = 190.766, 97.642, 75.078, 69.515; all P <0.001). The most common sites of MDRO infection were lower respiratory tract (48.39%), surgical site (11.83%), and urinary tract (11.02%). There were 641 cases of out-of-hospital infection (57.44%), including out-of-hospital transfer (33.42%) and community-acquire (24.01%), and 475 cases of nosocomial infection (42.56%), including our hospital transfer (4.66%) and department-acquire (37.90%). The composition of MDRO source was different among non-ICU departments. Conclusion: Nearly 2/3 of MDRO infection in non-ICU departments came from out-of-hospital input and in-hospital transfer. The implementation of basic infection control measures and early identification through information system need be noticed. The distribution and source of MDRO infection varied in different departments, and targeted prevention and control measures should be made according to its characteristics to achieve accurate prevention and control.