ObjectiveTo investigate the prevalence of healthcareassociated infection(HAI) in medical institutions in Pudong New Area. MethodsTen medical institutions in Pudong New Area were selected as monitored hospitals, crosssectional survey on HAI in each hospital at a given day of November 2013 and November 2014 were conducted. Prevalence of HAI in different levels of hospitals were compared. ResultsThe prevalence rates of HAI in  2013 and  2014 were 4.04% and 3.75% respectively, there was no significant difference between two years（χ2=0.709，P=0.400）. The prevalence rates of HAI in primary, secondary, and tertiary hospitals in 2013 were 0.66%,3.32%, and 4.60% respectively, in 2014 were 0, 3.52%, and 4.01% respectively，prevalence rates of HAI in different levels of hospitals of each year were significantly different （all P＜0.05）. Prevalence rates of HAI were high in departments of neurosurgery, hematology, intensive care units(ICUs), and gerontology. The prevalence rate of infection in ICUs increased obviously,from 10.09% to 18.78%（χ2=3.921，P=0.048），departments of gerontology decreased obviously, from 10.07% to 5.02%（χ2=5.698，P=0.017）.  The main HAI sites were lower respiratory tract (36.72%),  upper respiratory tract (9.96%), and urinary tract (12.89%). 172 pathogenic isolates were detected in 2013, and 177 were detected in 2014, gramnegative bacteria, grampositive bacteria, and fungi accounted for 60.74%,26.37%, and 12.89% respectively. Constituent ratios of pathogens causing HAI between two surveys were not significantly different (χ2=5.819，P=0.830）.ConclusionAmong different levels of hospitals in Pudong New Area, tertiary hospitals have the highest prevalence rate, HAI in ICU increases obviously, the main HAI site is lower respiratory tract, the main pathogens are gramnegative bacteria.