Background: Hepatitis B e Antigen (HBeAg) positive has been known as a risk factor for mother-to-child-transmission (MTCT) in addition to the high levels of hepatitis B virus (HBV) DNA. The World Health Organization (WHO) in 2020 recommends HBeAg testing as an alternative to determine eligibility for tenofovir prophylaxis, where molecular measurement is unavailable. However, high prevalence of HBeAg-negative chronic hepatitis B (e-CHB) in many countries should not be ignored. This study aimed to evaluate the viral characteristics in HBeAg-negative pregnant women and its association with the risk of intrauterine exposure to HBV. Methods: Antenatal screening of 1,348 pregnant women showed 81 (6·0%) HBsAg positivity. These HBsAg-positive mothers were examined for HBeAg status, HBsAg levels, viral load, and HBV DNA presence in corresponding cord blood. HBeAg-negative subjects were classified into inactive and ‘non-inactive’ carriers. Sequencing was performed to identify basal core promoter (BCP) and precore (PC) mutations in ‘non-inactive’ carriers. Findings: A total of 65 (80·2 %) of 81 HBsAg-positive subjects were HBeAg-negative. The risk for intrauterine exposure to HBV was higher at HBsAg levels ≥4 log 10 IU/mL (odds ratio [OR]=6·43; p =0·008), but comparable among the different HBeAg status. Higher risk of intrauterine exposure was found at HBV DNA levels ≥5·3 log 10 IU/mL (OR=22·32; p <0·001), with different risks for HBeAg-positive (OR=0·42; p =0·069) and HBeAg-negative (OR=12·6; p =0·010) groups. Of 65 HBeAg-negative women, 8 (13·8%) were inactive carriers and 57 (87·7%) were ‘non-inactive’ carriers. Ten of 12 HBV DNA sequences had BCP (A1762T/G1764A with or without T1753A/C), PC (A1814C or G1896A), or combined BCP/PC mutations, with higher proportion in genotype C than in genotype B. Interpretation: This study provided a cautionary note that HBeAg-negative status should be interpreted carefully, particularly for HBsAg-positive pregnant women who had high viral load. Funding Statement: Indonesia Science Fund and Educational Fund Management Institution (RISPRO/B1/TKL/5/6023/1/2017). Declaration of Interests: All authors declare to have nothing to disclose and have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest Ethics Approval Statement: This study was approved by the Research Ethics Committees of Hasanuddin University – Faculty of Medicine, Wahidin Sudirohusodo Hospital, and Hasanuddin University Hospital (Authorization No. 0942/H4.8.4.5.31/PP36-KOMETIK/2017).