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The characterization of mitochondrial genome has been evidenced as an efficient field of study for phylogenetic and evolutionary analysis in vertebrates including turtles. The aim of this study was to distinguish the structure and variability of the Trionychidae species mitogenomes through comparative analysis. The complete mitogenome (16796 bp) of an endangered freshwater turtle,  Nilssonia nigricans  was sequenced and annotated. The mitogenome encoded for 37 genes and a major non-coding control region (CR). The mitogenome was A + T biased (62.16%) and included six overlapping and 19 intergenic spacer regions. The Relative synonymous codon usage (RSCU) value was consistent among all the Trionychidae species; with the exception of significant reduction of Serine (TCG) frequency in  N. nigricans ,  N. formosa , and  R. swinhoei . In  N. nigricans , most of the transfer RNAs (tRNAs) were folded into classic clover-leaf secondary structures with Watson-Crick base pairing except for  trnS1  (GCT). The comparative analysis revealed that most of the tRNAs were structurally different, except for  trnE  (TTC),  trnQ  (TTG), and  trnM  (CAT). The structural features of tRNAs resulted ≥ 10 mismatched or wobble base pairings in 12 tRNAs, which reflects the nucleotide composition in both H- and L-strands. The mitogenome of  N. nigricans  also revealed two unique tandem repeats (ATTAT) 8,  and (TATTA) 20  in the CR. Further, the conserved motif 5′-GACATA-3′ and stable stem-loop structure was detected in the CRs of all Trionychidae species, which play an significant role in regulating transcription and replication in the mitochondrial genome. Further, the comparative analysis of Ka/Ks indicated negative selection in most of the protein coding genes (PCGs). The constructed Maximum Likelihood (ML) phylogeny using all PCGs showed clustering of  N. nigricans  with  N. formosa . The resulting phylogeny illustrated the similar topology as described previously and consistent with the taxonomic classification. However, more sampling from different taxonomic groups of Testudines and studies on their mitogenomics are desirable for better understanding of the phylogenetic and evolutionary relationships.

Complete mitochondrial genome of Black Soft-shell Turtle (Nilssonia nigricans) and comparative analysis with other Trionychidae