G-quadruplexes (G4) are four stranded nucleic acid secondary structures that form in vitro and in vivo in certain guanine rich sequences, upon stable non-Watson-Crick pairing of four guanines and stacking. G4 biological functions remain elusive, as it is difficult to identify the physiological and molecular circumstances of their formation, stabilization, recognition and processing. Inherently, biological processes generating single-stranded nucleic acids are favourable circumstances. Here, we questioned G4 formation during replication in the yeast S. cerevisiae and describe its pathological consequences. For this, we inserted the G-quadruplex-forming CEB1 minisatellite sequence near the strong and early firing origin ARS305 in both orientations and monitored its behaviour. We show that CEB1 tandem repeats are tolerated in wild-type cells but are very frequently destabilized upon treatment with the PhenDC3 G4-ligand and in the specific absence of the Pif1 unwinding helicase activity but in a preferential orientation manner, namely when the G-rich strand is template of leading strand replication. This instability is associated with the formation of Rad51-Rad52-dependent X-shaped molecules during replication, detected by 2D gels. This orientation-dependent destabilization of CEB1 relies on the presence of intact G4 motifs in CEB1, and on the activity of ARS305. The asymmetrical behavior of G4 prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G-rich telomeres.