By following the entire folding process (hours) of telomeric G4 at the single-molecule level and with a time resolution below 0.5 s, we could resolve several G4 states along the G4 folding pathway and provide a detailed description of their folding energetics and timescales. The combination of single-molecule experiments with different G4 forming DNA sequences and computational modeling of the experimental DNA constructs made possible the identification of several distinct folded G4 conformations of the human telomeric sequence that were not previously simultaneously observed. Single-molecule FRET is a powerful method for resolving several conformational states of complex biological systems that has been used for disentangling the structural polymorphism of telomeric G4s (14,49,52–54). However, previously, only two folded G4 states have been identified through single-molecule FRET experiments, which have been conditionally assigned to parallel and anti-parallel G4 conformations (14,49,52–54). In contrast, we identify FRET signatures of four distinct G4 conformations. The ability to directly resolve such large number of G4 conformations by FRET is not straightforward, due to similar size (Rg∼12 Å) (55) and comparable end-to-end distances for all G4 conformations. The resolution of different G4 conformations is achieved here through a careful design of the experimental system and choice of the specific positions for fluorophore labeling of the DNA constructs. Thus, our integrated approach, utilizing the power of single-molecule FRET microscopy and MD simulations, revealed the formation of two hybrid and two anti-parallel conformations in potassium containing solutions. One of the hybrid (hybrid 2) conformations and both anti-parallel conformations are kinetically favored during G4 folding, whereas the other hybrid conformation (hybrid 1) is the most thermodynamically stable structure for wild-type human telomeric G4s.