The recent introduction of entry inhibitors in the clinic as components of antiretroviral treatment has heightened the interest in coreceptor use of human immunodeficiency virus type 1 (HIV-1). Viruses using CCR5 as coreceptor (R5 viruses) are generally present over the entire course of infection whereas viruses using the CXCR4 coreceptor (R5X4/X4 viruses) emerge in about 50% of infected individuals during later stages of infection. The CCR5-to-CXCR4 switch represents a concern because CCR5 inhibitors, while suppressing R5 viruses, may allow the emergence of CXCR4-tropic viruses. In this study, HIV-1 populations that maintained CCR5 usage during infection were compared with populations that switched coreceptor usage to include CXCR4 later during infection, with the aim to find molecular properties of the virus populations associated with the CCR5-to-CXCR4 switch. We amplified and molecularly cloned the V1-V3 region of the HIV-1 envelope from 51 sequential HIV-1 isolates derived from 4 to 10 serial samples for each of the patients. Four of the patients had virus populations that switched coreceptor usage to include CXCR4 (switch populations: SP) during infection and four patients had viral populations that maintained exclusive CCR5 usage (non-switch populations: nSP). Coreceptor usage was determined experimentally on individual clones from dualtropic R5X4 isolates. In nSP we found that the number of potential N-linked glycosylation sites (PNGS) increased over time, whereas no pattern of change was observed in SP. We also found differences in V2 length and V3 charge between R5 viruses of nSP and R5 viruses of SP before the switch. The V2 region was significantly longer in R5 viruses of SP compared to viruses of nSP throughout the course of infection, and the V3 charge increased with time in R5 populations from SP, while it remained unchanged or decreased in nSP. These molecular properties could prove important for understanding the evolution of coreceptor usage in HIV-1 populations, and maybe even for predicting an upcoming coreceptor switch at early stages after primary infection.