In this study, we provide a detailed analysis of the kinetics and mutational pathways involved in the appearance of CXCR4-using variants during natural infection using V3 sequences generated by deep sequencing from PBMC and serum samples of eight HIV-1-infected individuals, in combination with V3-based coreceptor prediction tools, in the year before CXCR4-using variants were for the first time detected in the MT-2 assay. The gene discussed is CXCR4; the disease is infection.