Double-strand breaks (DSBs) are the most critical radiation-induced lesions, because they result in the fragmentation of the DNA molecule and because a single unrepaired DSB may lead to cell death. We present the results of radiation-induced fragmentation of plasmid DNA analyzed by atomic force microscopy (AFM) to allow the visualization of individual DNA molecules. Linear ΦX174 plasmid DNA was exposed to a wide range of doses of low-LET X rays and high-LET carbon, nickel and uranium ions. The induced DNA fragments were detected and measured based on the recorded AFM images and fragment length distributions were derived for each radiation type and dose. The results show a dose- and radiation type-dependent DNA fragmentation with a significantly larger fraction of short fragments produced by high-LET radiation compared to X rays. This can be considered as experimental evidence of DSB clustering due to inhomogeneous energy deposition at the level of the plasmid DNA molecule. Additionally, the experimentally derived fragment profiles were compared and found to be in agreement with the prediction of a model simulating the fragmentation of DNA molecules induced by radiation.