Difficulties to confirm and discriminate human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) infections by serological Western Blotting (WB) assay (HTLV Blot 2.4, MP Biomedicals) has been reported in Brazil, mainly in HIV/AIDS patients, with a large number of WB-indeterminate and WB-positive but HTLV untypeable results. Nonetheless, the line immunoassay (LIA) (INNO-LIA HTLV-I/II, Fujirebio) was pointed to enhance specificity and sensitivity for confirming HTLV-1/2 infections. To add information concerning the improved ability of LIA in relation to WB when applied in samples of individuals from different risk-groups from Brazil, we performed the present study. Three groups were analyzed: group 1 [G1], 62 samples from HIV/AIDS patients from São Paulo-SP (48 WB-indeterminate + 14 HTLV); group 2 [G2], 24 samples from patients with hepatitis B or hepatitis C from São Paulo (21 WB-indeterminate + 3 HTLV; 17 HIV-seropositive), and group 3 [G3], 25 samples from HTLV out-patients clinic from Salvador-Bahia (16 WB-indeterminate + 9 HTLV; all HIV-seronegative). Overall, the LIA confirmed HTLV-1/2 infection (HTLV-1, HTLV-2 or HTLV) in 66.1% [G1], 83.3% [G2], and 76.0% [G3] of samples. Interestingly, the majority of WB-indeterminate results were confirmed by LIA as HTLV-2 in G1 and G2, but not in G3, in which the samples were defined as HTLV-1 or HTLV positives. These results agree with the virus types that circulate in such patients of different regions in Brazil, and emphasize the LIA as the best serological test for confirming HTLV-1 and HTLV-2 infections, independently of being applied in HTLV-monoinfected or HTLV-coinfected individuals.