Using complementary molecular assays for DNA damage, telomere maintenance, and telomeric replication stress, we define the mechanisms underlying cytotoxicity induced by SMARCAL1 depletion in ALT-positive glioma cells.<h4>Results</h4>Our data demonstrate the annealing helicase SMARCAL1 is a highly specific synthetical lethal vulnerability in cancers that use ALT. Here, SMARCAL1 is linked to central nervous system cancer.