G-quadruplexes (G4s) are four-stranded guanine-rich structures that occur across the genome. They have been shown to play important regulatory roles during transcription and translation but can also act as a barrier during DNA replication. ATRX, a member of the SWI/SNF family of helicases, is known to bind to regions of the genome containing tandem repeats and high in GC-content (Law et al., 2010), suggesting that ATRX is involved in unwinding G4s. Corroborating this, cells lacking ATRX are more prone to cell death caused by G4 stabilising ligands (Wang et al., 2019). We have shown by immunofluorescence with a G4-specific antibody that there is increased G4 stabilisation across the genome in cell lines that use the Alternative Lengthening of Telomeres (ALT) mechanism for telomere maintenance, which often lack expression of ATRX, compared with those that use telomerase. G4 ChIP-seq analysis showed that while G4 formation is concentrated in the regulatory elements of genes in telomerase-positive (Tel+) cells, it is much more dispersed throughout the genome in ALT+ cells, suggesting a lack of regulatory function in the latter. Telomeric DNA is prone to formation of G4, so we postulated that deregulated telomeric G4 formation could contribute to telomere lengthening in ALT+ cells. Indeed, there was an increase in telomeric DNA synthesis outside S phase in ALT+ cells when treated with G4 stabilising ligands compared with untreated cells, indicative of ALT-mediated telomere lengthening. We postulate that deregulated G4 across the genome of ALT cells contributes to their high levels of genome instability. Taken together, our findings suggest a role for G4 in ALT-mediated telomere synthesis and a potential therapeutic avenue for G4 stabilising ligands in ALT cancers.