Adeno-associated virus (AAV) type 2 or UV-inactivated AAV (UV-AAV2) infection provokes a DNA damage response that leads to cell cycle arrest at the G2/M border. p53-deficient cells cannot sustain the G2 arrest, enter prolonged impaired mitosis, and die. Here, we studied how non-replicating AAV2 kills p53-deficient osteosarcoma cells. We found that the virus uncouples centriole duplication from the cell cycle, inducing centrosome overamplification that is dependent on Chk1, ATR and CDK kinases, and on G2 arrest. Interference with spindle checkpoint components Mad2 and BubR1 revealed unexpectedly that mitotic catastrophe occurs independently of spindle checkpoint function. We conclude that, in the p53-deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. As AAV2 acts through cellular damage response pathways, the results provide information on the role of Chk1 in mitotic catastrophe after DNA damage signaling in general.