Objective To investigate the current situation of healthcare-associated infection(HAI)in hospitals in Yunnan Province in 2015, and provide basis for formulating HAI control measures.Methods A cross-sectional survey on HAI prevalence rate , antimicrobial use,and pathogenic detection in 116 hospitals in Yunnan Province were conducted by combined methods of bed-side survey and medical record reviewing.Results A total of 64 386 hospitalized patients participated in the survey, the prevalence rate of HAI was 2.11%(n=1 357), and case prevalence rate was 2.29%(n=1 476).Difference in prevalence rates of HAI, antimicrobial usage rates, and pathogenic detection among hospitals of different beds were all significant(all P <0.01).The top 3 departments of HAI distribution were intensive care units(20.99%), burn departments(8.00%), and hematology departments (7.34%);the main infection site was lower respiratory tract(42.68%);HAI mainly caused by gram-negative bacteria(69.28%), the major were Escherichia coli , Klebsiella pneumoniae, and Pseudomonas aeruginosa.Antimicrobial usage rate at the survey day was 35.62 %, bacterial detection rate in patients receiving therapeutic antimicrobial use was 59.56%.Conclusion The cross-sectional survey on prevalence of HAI is helpful for understanding the current status of HAI, calculation of the percentile distribution of each index can promote self-evaluation for HAI-related work in each hospital.