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Summary: Background: Hepatitis B virus (HBV) is a gradually evolving virus. The aim of this study was to characterize the distribution pattern of HBV genotypes and subgenotypes and HBsAg subtypes in chronic hepatitis B subjects from the Indian subcontinent. We also sought to investigate the genetic diversity of HBV genotypes and its influence on the therapeutic response. Methods: A total of 295 chronic hepatitis B subjects were studied. HBV genotypes and subgenotypes were determined using the generated HBV reverse transcriptase (rt) sequences. HBsAg subtypes were predicted using a newly developed automated program in Microsoft Visual Basic (VB6). Genetic diversity was characterized by calculating the mean genetic distance (d), the number of synonymous substitutions per synonymous site (dS), and the number of non-synonymous substitutions per non-synonymous site (dN). The virological response was measured by HBV DNA levels. Results: In southern India, the predominant HBV subgenotype/subtype was D2/ayw3 (79.1%). In eastern India, C1/adr (28.2%) was found to be the predominant subgenotype/subtype, followed by A1/adw2 (25.4%). In the north-eastern region, C2/adr, D2/ayw3, and D5/ayw3 were predominant and were each identified in 20.8% of subjects. In treatment-naïve subjects, the d, dS, and dN of genotype D sequences were higher compared to genotypes C and A. Additionally, the d, dS, and dN of HBV rt sequence were higher in subjects who subsequently showed a virological response to nucleos(t)ide analogues as compared to non-responders, irrespective of the genotypes tested (p =0.014 to p <0.0001). Conclusions: We have described the distribution of HBV genotypes and subgenotypes and HBsAg subtypes in three major regions of the Indian subcontinent. HBV genetic diversity may play a pivotal role in the clinical outcome of chronic hepatitis B. [ABSTRACT FROM AUTHOR]