Different strategies can be applied for the screening of HIV infection, depending on the local seroprevalence. Within a WHO type III strategy, we compared the results of two different second-line methods for HIV screening of a population of pregnant women in the Republic of Congo. Sera from 3614 consecutive pregnant women were tested for HIV with Genescreen Plus Ag/Ab EIA assay; positive specimens were retested with two different second-line methods. (Determine HIV-1/2 rapid test and Vironostika HIV Ag/Ab specific EIA assay). Discordant samples were tested with HIV-1/2 Western Blot and, if necessary, HIV RNA molecular assay. Of the 3614 sera, 221 were positive with Genscreen. Among them, 21 and 10 tested negative with Vironostika and Determine, respectively. A 100% correspondence with 3rd line confirmation test results was found in Genscreen positive/Vironostika negative samples, whereas a 5.5% overestimation of HIV seroprevalence was observed when Determine, instead of Vironostika, was used as second-line test. The choice of appropriate assays in adequate sequence, within the correct WHO strategy, is pivotal to minimize the risk of overtreatment of HIV infection.