Cancers that utilize the Alternative Lengthening of Telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of PML nuclear bodies (PML NBs) which are required for intrinsic immunity to various viruses. Here we asked whether ATRX-deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in killing ATRX-deficient cells. Infection of co-cultured primary and ATRX-null cancer cells revealed that mutant HSV-1 selectively killed ATRX-null cells. Sensitivity to mutant HSV–1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.