HIV-1 RNA level is strongly associated with HIV transmission risk. We sought to determine whether HIV-1 RNA level was associated with prior knowledge of HIV status among treatment-naive HIV-infected individuals in Botswana, a country with high rates of antiretroviral treatment (ART) coverage. This information may be helpful in targeting HIV diagnosis and treatment efforts in similar high HIV prevalence settings in a population-based survey. HIV-infected individuals were identified during a household survey performed in 30 communities across Botswana. ART-naive persons with detectable HIV-1 RNA (>400 copies/mL) were divided into two groups, newly diagnosed and individuals tested in the past who knew about their HIV infection at the time of household visit, but had not taken ART. Levels of HIV-1 RNA were compared between groups, overall and by age and gender. Among 815 HIV-infected ART-naive persons with detectable virus, newly diagnosed individuals had higher levels of HIV-1 RNA (n = 490, median HIV-1 RNA 4.35, interquartile range (IQR) 3.79-4.91 log10 copies/mL) than those who knew about their HIV-positive status (n = 325, median HIV-1 RNA 4.10, IQR 3.55-4.68 log10 copies/mL; p values <.001, but p value = .011 after adjusting for age and gender). A nonsignificant trend for higher HIV-1 RNA was found among newly diagnosed men 30 years of age or older (median HIV-1 RNA 4.58, IQR 4.07-5.02 log10 copies/mL vs. 4.17, 3.61-4.71 log10 copies/mL). Newly diagnosed individuals have elevated levels of HIV-1 RNA. This study highlights the need for early diagnosis and treatment of HIV infection for purposes of HIV epidemic control, even in a setting with high ART coverage.Read less <