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The environmental conditions required by many extremophiles for their optimal growth raise questions about these organisms’ ability to replicate their chromosomal DNA accurately. To address this question for a halophilic archaeon, Haloferax volcanii and a thermophilic bacterium, Thermus thermophilus a quantative assay of spontaneous mutation rate was developed using 5 - fluoro orotic acid (FOA) selection. In Haloferax volcanii, the estimated spontaneous mutation rate using fluctuation assays was 2.42 x 10 -8 +/- 1.4x10 -8 mutational events per cell per division cycle and in Thermus thermophilus the rate was 2.98 x 10 -7 +/- 2.70 x 10 -7 mutational events per cell per division cycle for pyrE and pyrF genes. These mutation rates are similar to or lower than those of non-extremophiles. Thus these studies have shown that extremophiles maintain high genetic fidelity even though they grow in extreme environments. These facts are useful in exploring the issues related to DNA repair in archaea and bacteria found in extreme habitats.