Highly active antiretroviral therapy (HAART) can suppress plasma human immunodeficiency virus type 1 (HIV-1) levels to below the detection limit of ultrasensitive clinical assays. However, HIV-1 persists in cellular reservoirs, and in adults, persistent low-level viremia is detected with more sensitive assays. The nature of this viremia is poorly understood, and it is unclear whether viremia persists in children on HAART, particularly those who start therapy shortly after birth. We therefore developed a reverse transcriptase PCR (RT-PCR) assay that allows genotyping of HIV-1 protease even when viremia is present at levels as low as 5 copies of HIV-1 RNA/ml. We demonstrated that viremia persists in children with plasma virus levels below the limit of detection of clinical assays. Viremia was detected even in children who began HAART in early infancy and maintained such strong suppression of viremia that HIV-1-specific antibody responses were absent or minimal. The low-level plasma virus lacked protease inhibitor resistance mutations despite the frequent use of nelfinavir, which has a low mutational barrier to resistance. Protease sequences resembled those of viruses in the latent reservoir in resting CD4⁺ T cells. Thus, in most children on HAART with clinically undetectable viremia, there is continued virus production without evolution of resistance in the protease gene.