AbstractThe mitochondrial genome of a golden orb-web spiderTrichonephila clavata(L. Koch, 1878) from South Korea is determined and characterized in detail, which is the second mitochondrial genome reported from this species: the first was published from the Chinese sample by Pan et al. (2016). It was 14,436 bp in length being composed of 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and one control region (CR). It has a base composition of 35.99% for ‘A,’ 14.88% for ‘G,’ 9.09% for ‘C,’ and 40.04% for ‘T.’ Comparing the South Korean and Chinese mitochondrial genomes, we observed 8% nucleotide sequence differences between their CRs, caused by the different numbers and sorts of possessed tandem repeats, suggesting a promising molecular marker to distinguish South Korean individuals from Chinese ones. The phylogenetic trees using the maximum likelihood (ML) method were reconstructed with nucleotides (without 3rd codon position) and amino acids from 13 PCGs, respectively, which consistently confirmed thatT. clavata(Subfamily Nephilinae) from South Korea and China are clustered together, distinctly separated from the other subfamily Araneinae in the monophyletic family Araneidae.