The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 105 to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r2 = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient. Copyright © 2009, American Society for Microbiology. All Rights Reserved.