Background/Aims: Acute exacerbations of chronic hepatitis B (CHB-AEs) are common in endemic areas and are often presumed to be acute hepatitis B (AHB) due to their similarities in clinical and serological pictures, presenting a major diagnostic dilemma. This study aimed to identify laboratory markers for differentiating between the two groups, and to establish the cut-off value for significant markers. Methods: A retrospective analysis of records was conducted for patients who presented with clinical features of acute hepatitis along with hepatitis B surface antigen (HBsAg) and IgM antibody to hepatitis B core antigen (IgM anti-HBc) positivity from May 2015 to May 2017. A total of 172 patients were enrolled and grouped as AHB (n=89) and CHB-AE (n=83) based on their history of hepatitis B virus infection and duration of HBsAg persistence. Virological and biochemical parameters were analyzed and compared. Cut-off values, sensitivity, and specificity of the variables were calculated. Results: The median value of signal by cut-off (S/Co) ratio for IgM anti-HBc was significantly higher in AHB group (30.44) compared to CHB-AE group (8.63) with a sensitivity and specificity of 97% and 84%, respectively, at a cut-off of 20.5 (P<0.01). The mean international normalized ratio (INR) was significantly greater in CHB-AE (1.88±1.24) group compared to AHB group (1.62±0.17) with a sensitivity and specificity of 57.9% and 45.1%, respectively, at a cut-off value of 1.27. Conclusions A value of 20.5 S/Co of IgM anti-HBc and 1.27 INR could be helpful in differentiating between AHB and CHB-AE.