[Objectives] In China, the genetic resources of Chinese Soft-shelled Turtle(Pelodiscus sinensis) are diverse, encompassing various geographical populations, aquaculture stocks, and distinct breeding lines. Differences are evident not only between wild specimens caught from different regions but also among artificially selected strains, particularly in terms of productivity, body morphology (including marginal flap size), and other traits. Consequently, distinguishing specific genetic lineages by a single species is challenging. Currently, efforts to conserve the genetic resources of P. sinensis and breed new varieties (strains) are advancing in an orderly manner. P. sinensis “Poyang Lake No. 1” (provisionally named) is a new strain bred by our team, originating from the Poyang Lake region of China. It was developed through five consecutive generations of mass selection, with growth rate and marginal flap width as the target traits. The strain now exhibits markedly improved performance, including accelerated growth, broad marginal flaps, and elevated egg production. This study aimed to: 1) characterize the complete mitochondrial genome of this elite strain to elucidate its unique molecular features; 2) determine its precise phylogenetic position among various P. sinensis populations/strains; and 3) identify potential mitochondrial DNA markers for accurate germplasm identification of this strain. [Methods] The complete mitochondrial genome sequence of the F5 generation of “Poyang Lake No. 1” was obtained by PCR amplification with 17 specifically designed primer pairs (Table 1) covering the entire genome and Sanger sequencing. Bioinformatics tools (GeSeq, tRNAscan-SE, Tandem Repeats Finder, SnapGene, Editseq7.1, GB2Sequin, and OGDraw) were used for structural annotation, compositional analysis, and repeat identification of the assembled sequence (GenBank accession number:PV472338). Phylogenetic relationships were reconstructed in MEGA 11.0 based on two datasets:1) concatenated nucleotide sequences of 12 heavy-strand protein-coding genes (PCGs-H); and 2) the cytochrome b (Cyt b) gene sequence. The analysis included “Poyang Lake No. 1” and eight other reference strains/populations, with Palea steindachneri as the outgroup. Trees were constructed via the Neighbor-Joining (NJ) method. [Results] The complete mitochondrial genome of “Poyang Lake No.1” was 17 190 bp in length (Fig. 2). It exhibited a significant AT bias, with nucleotide composition on the heavy strand (H-strand) as follows:35.47% adenine (A), 27.34% thymine (T), 25.51% cytosine (C), and 11.68% guanine (G), and based on this composition, the AT content of the H-strand reached 62.81%. A total of 37 genes were annotated:13 protein-coding genes (PCGs), 22 tRNA genes (including two copies of tRNA-Ser and tRNA-Leu), and 2 rRNA genes. With the exception of 8 tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro) and the ND6 gene located on the light strand (L-strand), all the other genes were encoded by the H-strand (Table 3). The gene order was identical to that of other reported P. sinensis strains and most vertebrates. The control region (D-loop) was notably long (1 666 bp) and contained four tandem repeat units, including a 50-bp motif repeated 6 times. Sequence length variations among different P. sinensis strains were primarily concentrated in the D-loop region, ranging from -151 bp to +164 compared with that of P. sinensis “Poyang Lake No. 1” (Table 2). An extra T base insertion was identified at position 169 of the mitochondrial ND3 gene in “Poyang Lake No. 1”. Phylogenetic analyses based on both PCGs-H concatenated sequences and the Cyt b gene yielded congruent topologies (Fig. 3). The results clearly demonstrated that P. sinensis “Poyang Lake No.1” and the P. sinensis “Japanese” strain formed a closely related clade, which then constituted a sister branch with the P. sinensis “Korean” strain. Five other Chinese strains/populations (“Anhui Wuhu”, “Anhui Inked”, “Jiangxi”, “Qingxi Black”, and “Qingxi Variegated”) clustered together in a separate, well-defined clade. [Conclusion] This study provides the first complete mitochondrial genome sequence of the selectively bred P. sinensis strain “Poyang Lake No. 1”. Key characteristics include its distinct D-loop length and repeat structure, specific nucleotide composition, and the unique ND3 insertion.Phylogenetic analysis robustly demonstrates that “Poyang Lake No. 1” shares its most recent common ancestor with the “Japanese” strain, forms a sister group with the “Korean” strain, and is genetically distinct from major strains found in the Yangtze River basin. The identified features, particularly the D-loop structure, PCGs-H haplotypes, and Cyt b sequence variations, serve as highly effective molecular markers for the precise identification and traceability of the “Poyang Lake No. 1” germplasm, supporting its conservation and breeding management.