The complete mitogenomes of two orb-weaving spiders Neoscona doenitzi and Neoscona nautica were determined and a comparative mitogenomic analysis was performed to depict evolutionary trends of spider mitogenomes. The circular mitogenomes are 14,161bp with A+T content of 74.6% in N. doenitzi and 14,049bp with A+T content of 78.8% in N. nautica, respectively. Both mitogenomes contain a standard set of 37 genes typically presented in metazoans. Gene content and orientation are identical to all previously sequenced spider mitogenomes, while gene order is rearranged by tRNAs translocation when compared with the putative ancestral gene arrangement pattern presented by Limulus polyphemus. A comparative mitogenomic analysis reveals that the nucleotide composition bias is obviously divergent between spiders in suborder Opisthothelae and Mesothelae. The loss of D-arm in the trnSUCN among all of Opisthothelae spiders highly suggested that this common feature is a synapomorphy for entire suborder Opisthothelae. Moreover, the trnSAGN in araneoids preferred to use TCT as an anticodon rather than the typical anticodon GCT. Phylogenetic analysis based on the 13 protein-coding gene sequences consistently yields trees that nest the two Neoscona spiders within Araneidae and recover superfamily Araneoidea as a monophyletic group. The molecular information acquired from the results of this study should be very useful for future research on mitogenomic evolution and genetic diversities in spiders. •The complete mitogenomes of two Neoscona spiders contain a standard set of 37 genes and a control region.•The nucleotide composition bias is obviously divergent between spiders in suborder Opisthothelae and Mesothelae.•The loss of D-arm in the trnSUCN is a synapomorphy for entire suborder Opisthothelae.•Phylogenetic analysis indicates that mitogenome sequences are useful in resolving higher-level relationship of Araneae