Significance HIV-1 infection requires lifelong treatment with antiretroviral therapy (ART) due to viral rebound of a latent reservoir of intact, transcriptionally silent provirus found to persist in the genome of CD4 ⁺ T cells. One major challenge to understanding the nature of the latent reservoir is accurately characterizing the measuring of the size of the reservoir. Herein, we use quadruplex polymerase chain reaction (Q4PCR) to assess the dynamics of the latent reservoir in HIV ⁺ individuals who have been on long-term ART for up to 10 y. Our results show that Q4PCR can be used to accurately measure the latent reservoir, while providing the added benefit of assessing the genetic diversity of the reservoir to better understand changes to clonal dynamics over time.