TEMPO and its derivatives are stable free radical nitroxides widely used in the field of chemistry, biology, and pharmacology. TEMPO was previously found to be mutagenic and to induce micronuclei in mammalian cells. In the present study, we investigated and quantified the genotoxicity of four structurally similar nitroxides, TEMPO and three of its derivatives (4-hydroxy-TEMPO, 4-oxo-TEMPO, and 4-methoxy-TEMPO), using the mouse lymphoma assay (MLA) and Comet assay in L5178Y Tk+/- cells. The results showed that all tested nitroxides were cytotoxic and mutagenic in the MLA, both in the presence and absence of S9, with metabolic activation significantly enhancing the cytotoxicity and/or mutagenicity. In addition, the four nitroxides caused DNA strand breakage. The mutagenicity and DNA damaging dose-responses of the test articles were compared using the PROAST benchmark dose (BMD) software package. The potency ranking of the four nitroxides for mutagenicity was different from the ranking of the DNA damaging effects. The mode of action (MoA) analysis by a multi-endpoint DNA damage pathway assay classified all four nitroxides as clastogens. In addition, the majority of the induced Tk mutants showed loss of heterozygosity (LOH) at the Tk and D11Mit42 loci (i.e., chromosome damage <31 Mbp). These results suggest that TEMPO and its three derivatives are cytotoxic and mutagenic in mouse lymphoma cells through a mechanism that involves strand breakage and large alterations to DNA. The potency rankings indicate that the different TEMPO derivatives vary in their mutagenic and DNA damaging potential.