In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single-and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (⌬pyrF) back-ground, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophi-lus was inserted into the chromosome of UBOCC-3256 (⌬pyrF) or UBOCC-3260 (⌬pyrF ⌬hisB), allowing homologous comple-mentation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.