The polymorphisms of IFNL3 associated with the outcomes of primary HBV infection remains controversial, the susceptibility has not been investigated across cohorts. So two cohorts, including 3874 participants, were enrolled to detect the association of IFNL3 genetic variants (rs12979860, rs12980275, and rs809917) with outcomes of primary HBV infection between groups of chronic HBV infection (CHB), Natural clearance (NC), Health control (HC), and a false-positive report probability (FPRP) test was applied to assess positive results, and function prediction of significant polymorphism was evaluated. Polymorphism of rs12979860 demonstrated a correlation to the outcomes of primary infection. In comparison between NC and CHB, the rs12979860-C significantly decreased the risk of CHB as compared to T allele in Hubei, Hainan and combined two cohorts, and in HC vs. NC comparison, the allele of rs12979860-C also refers immunity establishment against HBV in comparison to T allele in Hubei, Hainan, and combined two cohorts. In comparison between NC and CHB, the rs12979860-TT genotypes significantly increased the risk of CHB in Hubei, Hainan and combined two cohort. After adjusting the influence of age and sex, the results remain significant. FPRP test confirms the significance of rs12979860, moreover, rs12979860-C/T alteration brings the minimum free energy change in the centroid secondary structure. The polymorphism of rs12979860 in IFNL3 is associated with primary outcomes of HBV infection, and rs12979860-CC is associated with immunity establishment in primary HBV infection, serving as potential predictors against virus infection.