Objective To investigate the clinical features of acute hepatitis B (AHB) and acute exacerbation of chronic hepatitis B (CHB) for differential diagnosis. Methods A retrospective analysis was performed on the clinical data of 96 AHB patients and 124 patients with acute exacerbation of CHB, who were admitted to the Public Health Clinical Center Affiliated to Fudan University from June to December, 2014. Comparison of continuous data between the two groups was made by Mann Whitney U test, while comparison of categorical data was made by chi-square test. Results There were no significant differences in the age of onset and sex between the AHB group and the acute exacerbation of CHB group; the incidence was higher in males than in females. Sexual transmission and iatrogenic transmission were the main routes of transmission for AHB, while mother-to-child transmission was the main route of transmission for acute exacerbation of CHB. The sensitivity and specificity of alanine aminotransferase (ALT) level ≥1072 U/L for diagnosing AHB were 78.6% and 79.2%, respectively. The sensitivity and specificity of S/CO ≥13.6 in the anti-HBc-IgM test for diagnosing AHB were 94.5% and 89.3%, respectively. At week 2 after admission, the AHB group showed significantly greater decreases in levels of HBsAg, HBeAg, and hepatitis B virus (HBV) DNA than the acute exacerbation of CHB group (P＜0.05). At week 8 after admission, the AHB group had significantly higher HBsAg clearance rate, anti-HBs seroconversion rate, HBeAg clearance rate, anti-HBe seroconversion rate, and HBV DNA clearance rate than the acute exacerbation of CHB group (P＜0.05). ConclusionIt is helpful for making the differential diagnosis between AHB and acute exacerbation of CHB to know the route of transmission, ALT level, anti-HBc-IgM test result (S/CO value), HBV DNA clearance rate, and the seroconversion rates of HBV markers. [ABSTRACT FROM AUTHOR]