Restriction endonucleases (REases) are enzymes that recognize and cleave DNA in a sequence specific manner. The recognition site consists of a sequence of nucleotides in the DNA duplex, typically four to eight base pairs long. Most of the commercially produced REases are isolated from the mesophilic bacteria. But the disadvantage of REases from mesophilic sources is that these enzymes are usually denatured at ambient and high temperature. As temperature produces opposite effects on both enzyme activity and stability, it is therefore a key variable in any biocatalytic process. Also, mesophilic enzymes are unstable, have low reactivity, lose activity during purification, and require refrigerated transport and storage. So, thermostable REases are preferred to circumvent these problems. This chapter deals mainly with thermophilic REases. The increasing interest in this field is reflected by the growing information on the discovery, purification, and characterization of REases from thermophilic sources. The properties associated with these enzymes offer additional advantages over their mesophilic counterparts.