Current laboratory methods to detect recent HIV-1 infection for the estimation of incidence have various limitations, including varying performance in different subtypes or populations. Therefore, new methods are needed to detect recent infections with increased specificity. We developed a recombinant protein, rIDR-M, that covered divergent sequences from the immunodominant region (IDR) of gp41 from all major subtypes and recombinants of HIV-1 group M and expressed in Escherichia coli. The rIDR-M protein was highly reactive with HIV antibodies in sera from different subtypes and equivalently detected antibodies to divergent subtypes B and AE from Thailand, in contrast to individual gp41 peptides derived from respective subtypes, suggesting that it can be used for incidence assays. The protein was used in two different assay formats to measure antibody avidity: (1) a two-well avidity index assay (AI-EIA) and (2) a new one-well limiting antigen avidity assay (LAg-avidity EIA), both with a pH 3.0 buffer to dissociate low-avidity antibodies present during early infection. Limiting the amount of antigen allowed detection of recent HIV-1 infection, with or without dissociation buffer, but the detection was most efficient when the pH 3.0 dissociation buffer was included. When a well-characterized 41-member seroincidence panel (20 recent and 21 long-term) was used, both the two-well AI-EIA and one-well LAg-avidity EIA efficiently distinguished recent and long-term infections. The new avidity-based assays using rIDR-M antigen may improve the accuracy of detecting recent HIV-1 infection and allow a better estimation of incidence in diverse HIV-1 subtypes.