Influenza-like illness (ILI) is an acute respiratory infection caused by various pathogens. However, the epidemiologic characteristics of ILI pathogens in Jiangsu province are unclear. To better understand the ILI etiology, the characteristics of the pathogens from nasopharyngeal swab samples of patients with ILI collected from 2012 to 2016 in 6 hospitals in Jiangsu province were studied. The pathogens, including influenza virus, respiratory syncytial virus (RSV), rhinovirus (HRV), adenovirus (ADV), herpes simplex virus (HSV), human coronavirus (hCoV), Streptococcus pneumoniae and Haemophilus influenzae, were detected by real-time PCR. At least one pathogen was identified in 1 334 of the patients (40.23%). Among viruses, HRV, influenza A virus (Flu A), ADV and RSV were the most frequently detected. ADV was the only pathogen that was distributed evenly in different years and regions (P>0.05). The etiological distribution varied in different age groups. Streptococcus pneumoniae was the most common pathogen in co-infections with a co-detection rate of 64.57% (319/494). The spectrum of etiologies could help to estimate disease burden and provide guidance for vaccination.