Transcription-mediated amplification assays for HBV DNA detection have transitioned from the Ultrio to the Ultrio Plus assay, which features increased analytic sensitivity due to inclusion of a target enhancer reagent. The impact on HBV detection for different categories of HBV infection has not been fully evaluated. Hepatitis B virus (HBV) DNA and hepatitis B surface antigen (HBsAg) detection rates as well as viral load (VL) distributions in HBV nucleic acid test (NAT)-yield samples were compared during 1 year of screening of South African blood donors with the Ultrio assay and the subsequent year by the Ultrio Plus version. HBV-DNA concentration at the HBsAg seroconversion point was established by regression analysis using a set of antibody to hepatitis B core antigen-negative acute viremic samples. Ultrio Plus detected twofold more window-period (WP) NAT yield donations and 1.7-fold more occult HBV infections than Ultrio. The VL distribution data indicated that Ultrio not only missed samples of less than 100 copies/mL, but also a substantial number higher than this level. The VL at the HBsAg seroconversion point was estimated at 916 copies/mL, whereas the VL at the NAT-conversion points was calculated at 63 and 4.1 copies/mL for Ultrio and Ultrio Plus. This reduced the infectious WP (compared to HBsAg testing) by 10.3 and 20.4 days, respectively. The higher-than-expected increase in HBV-NAT yields after introduction of the Ultrio Plus assay is likely attributable to variable sensitivity of the former Ultrio assay for different HBV samples. Therefore, previously published HBV WP reduction and residual risk estimates based on analytical sensitivity of the Ultrio assay need to be revised. © 2019 AABB.