SummaryBackgroundAn accurate, single‐point differential diagnosis between HBeAg‐negative infection (ENI) and chronic hepatitis B (CHB) is an unmet need.AimsTo assess the diagnostic value of the new hepatitis B core‐related antigen (HBcrAg) assay.MethodsA retrospective anonymised data analysis was performed in a multicentre European (nine centres and six countries) cohort of 1582 consecutive HBsAg‐positive/HBeAg‐negative subjects classified according to EASL guidelines as: 550‐CHB, 710‐ENI and 322‐GZ (grey‐zone, HBV‐DNA <20 000 IU/mL).ResultsMean age was 44 (±13.2 y), 59% were men; HBV genotypes were 15% A, 2% B, 2% C, 45% D, 9% E, 1% F and 26% unknown. Median HBV‐DNA serum levels were 2.2 (1.5‐2.7), 3.5 (3.2‐3.8) and 5.6 (4.8‐6.6) logIU/mL in ENI, GZ and CHB, P < 0.0001. HBsAg serum levels (HBsAgsl) were comparable in CHB and GZ, but lower in ENI (2.9 [2.1‐3.6] logIU/mL), P < 0.0001. HBcrAg serum levels (HBcrAgsl) were <3 logU/mL in 90.7% (644/710) ENI, 75.2% (242/322) GZ and 4.7% (26/550) CHB (P < 0.0001). Median HBcrAgsl were 4.8 (3.9‐5.7), 2.5 (2.0‐2.9) and 2.0 (2.0‐2.5) logU/mL in CHB, GZ and ENI, (P < 0.0001). ROC‐AUCs for HBcrAg and HBsAg were 0.968 (95% CI, 0.958‐0.977) and 0.732 (95% CI, 0.704‐0.760) respectively. The optimal HBcrAgsl cut‐off to distinguish CHB from ENI was 3.14 logU/mL (95% CI, 3.02‐3.25, 91% SE, 93% SP and 92.4% DA). HBcrAgsl were associated with HBV genotypes (P < 0.001, one‐way ANOVA) but using genotype‐specific cut‐offs, HBcrAg DA remained unchanged with overlapping 95% CI.ConclusionThe HBcrAg assay showed high diagnostic performance in the accurate single‐point identification of patients with HBeAg‐negative CHB, independently of HBV genotype. This should prompt future prospective studies to confirm its diagnostic role in clinical practice.