Abstract About 10–15% of all human cancer cells employ a telomerase-independent recombination-based telomere maintenance method, known as alternative lengthening of telomere (ALT), of which the full mechanism remains incompletely understood. While implicated in previous studies as the initiating signals for ALT telomere repair, the prevalence of non-canonical nucleic acid structures in ALT cancers remains unclear. Extending earlier reports, we observe higher levels of DNA/RNA hybrids (R-loops) in ALT-positive (ALT+) compared to telomerase-positive (TERT+) cells. Strikingly, we observe even more pronounced differences for an associated four-stranded nucleic acid structure, G-quadruplex (G4). G4 signals are found at the telomere and are broadly associated with telomere length and accompanied by DNA damage markers. We establish an interdependent relationship between ALT-associated G4s and R-loops and confirm that these two structures can be spatially linked into unique structures, G-loops, at the telomere. Additionally, stabilization of G4s and R-loops cooperatively enhances ALT-activity. However, co-stabilization at higher doses resulted in cytotoxicity in a synergistic manner. Nuclear G4 signals are significantly and reproducibly different between ALT+ and TERT+ low-grade glioma tumours. Together, we present G4 as a novel hallmark of ALT cancers with potential future applications as a convenient biomarker for identifying ALT+ tumours and as therapeutic targets.