Background and ObjectivesSerological HTLV‐1/2 screening is mandatory for blood donor candidates in Brazil. Our objective was to analyse HTLV test results in blood donors submitted for screening and confirmatory assays in a Brazilian blood bank.Materials and MethodsRetrospective analysis (2017–2022) results of chemiluminescence immunoassays and confirmatory tests for HTLV‐1/2 in reactive donors were performed. During the analysed period, three sets of assays were used: (1) Architect rHTLV‐I/II + HTLV Blot 2.4 (Western blot [WB]); (2) Alinity s HTLV I/II Reagent Kit + INNO‐line immunoassay (LIA) HTLV I/II Score (LIA); (3) Alinity + WB.ResultsThe analysed period comprised a total of 1,557,333 donations. The mean percentage of HTLV reactive donors using the Architect assay was 0.14%. With the change to the Alinity assay, that percentage dropped 2.3‐fold (0.06%). The reactivity rate in the confirmatory tests (1064 samples) ranged from 13.5% to 30.2%, whereas 58.3%–85.9% of samples were non‐reactive. The highest rates of positive (30.2%) and indeterminate (11.5%) results were seen using LIA. Considering all analysed samples, those with signal/cut‐off ratio (S/CO) >50 were positive in confirmatory tests (positive predictive value, PPV = 100%), whereas samples with S/CO ≤6 are very unlikely to be truly positive (PPV = 0).ConclusionThe use of the Alinity assay reduced the frequency of false‐positive results. Confirmatory tests are important to identify true HTLV infection in blood donors, because more than 58% of initially reactive individuals are confirmed as seronegative. Categorizing S/CO values is useful for assessing the likelihood of true HTLV‐1/2 infection.