Objective:To evaluate the detection accuracy of hepatitis B virus (HBV) serological markers among grassroots medical institutions in the demonstration areas of infectious diseases.Methods:A fixed sampling method was used among the followed-up hepatitis B cohort in general population of six infectious disease demonstration areas nationwide. Blood samples of chosen objects were collected, in which HBsAg and anti-HBs were tested by grassroots medical institutions and were also parallely tested by the third-party platform. The test results were compared between the two groups. Statistical analyses were conducted by SAS 9.4 software.Results:A total of 5 756 and 5 263 samples of HBsAg and anti-HBs were successfully tested, respectively. Comparing the results of HBsAg and anti-HBs from grassroots medical institutions with the results from the third platform, the agreement of HBsAg and anti-HBs was 97.13% and 77.33%, respectively. The Kappa value was 0.56 (95% CI 0.50-0.62) for HBsAg and 0.54 (95% CI 0.52-0.56) for anti-HBs, respectively; and the McNemar tests indicated the difference between the results (all P<0.01). There were also significant differences in agreement of testing results with the third platformin among different regions ( P<0.05 or <0.01). The Kappa values indicated that Jiangsu province and Guangdong province had high accordance rates of HBsAg (0.87 and 0.81, respectively), and Gansu province and Guangdong province had high accordance rates of anti-HBs (Both were 0.74). Regarding the results from the third platform as the standard, the sensitivity of HBsAg testing in grassroots medical institutions was moderate (40.51%) and the specificity was well (99.96%). The sensitivity of anti-HBs testing was substantial (73.18%) and the specificity was well (84.31%). Guangdong province (Youden index: 0.69) and Jiangsu province (Youden index: 0.80) had high identification ability for HBsAg, and for indicator of anti-HBs, Gansu province (Youden index: 0.78) and Guangdong province (Youden index: 0.76) had high identification ability. Conclusion:There are certain differences in results of HBV serological markers tests between the grassroots medical institutions in the demonstration areas of infectious diseases and the third platform. Current testing strategies in grassroots medical institutions are suitable for identifying people without hepatitis B infection, while it is necessary to pay attention to the situation of potential false negative error.