Hepatitis B virus (HBV) genotyping plays an important role in the clinical management of chronic hepatitis B (CHB) patients. However, the current nucleic acid based techniques are expensive, time-consuming, and inconvenient. Here, we developed a novel DNA-independent HBV genotyping tool based on a one-step fluorescent lateral flow immunoassay (LFIA). Epitope-targeting immunization and screening techniques were used to develop HBV genotype specific monoclonal antibodies (mAbs). These mAbs were used to develop a multitest LFIA with a matched scanning luminoscope for HBV genotyping (named the GT-LFIA). The performance of this novel assay was carefully evaluated in well-characterized clinical cohorts. The GT-LFIA, which can specifically differentiate HBV genotypes A, B, C, and D in a pretreatment-free single test, was successfully developed using four genotype specific mAbs. The detection limits of the GT-LFIA for HBV genotypes A, B, C, and D were 2.5-10.0 IU HBV surface antigen/mL, respectively. Among the sera from 456 CHB patients, 439 (96.3%; 95% confidence interval (CI), 94.1-97.8%) were genotype-differentiable by the GT-LFIA and 437 (99.5%; 95% CI, 98.4-99.9%) were consistent with viral genome sequencing. In the 21 patients receiving nucleos(t)ide analogue therapy, for end-of-treatment specimens that were HBV DNA undetectable and were not applicable for DNA-dependent genotyping, the GT-LFIA presented genotyping results that were consistent with those obtained in pretreatment specimens by viral genome sequencing and the GT-LFIA. In conclusion, the novel GT-LFIA is a convenient, fast, and reliable tool for differential HBV genotyping, especially in patients with low or undetectable HBV DNA levels.