In order to investigate the biodiversity of amphibian and reptile in the Nanshan National Park, Chengbu County, Hunan Province, four specimens of the Bufo genus were collected (specimen numbers:HNNU2024032401﹣HNNU2024032404) near a stream in the Jintongshan area (26°09′12″ N, 110°09′17″ E, altitude 1 760 m) in April 2024.The specimens were identified based on morphological characteristics described by Nihn et al. (2022) and Fei et al. (2009). Two mitochondrial genes, namely 16S ribosomal RNA gene (16S rRNA) and cytochrome C oxidase I gene (COI), were concatenated into a single sequence of 1 140 bp for phylogenetic analysis. Other sequences were obtained from GenBank (Appendix 1). DNA sequences were aligned in MEGA 7 by the Clustal W algorithm with default parameters. The dataset was partitioned according to genes and codons for 16S rRNA and COI, then tested in PhyloSuite 1.2.2, resulting in the best-fitting nucleotide substitution models of TIM2 + I + G. Sequenced data were analyzed using Bayesian inference (BI) and maximum likelihood (ML) approaches. Two independent runs were conducted in the BI analysis with 2 000 000 generations each, and sampled every 1 000 generations with the first 25% of samples discarded as burn-in. In the ML analysis, a bootstrap consensus tree was generated from 1 000 replicates. Both the ML and BI analyses yielded essentially identical topologies, pairwise distances were calculated in MEGA 7 using the uncorrected model based on 16S rRNA sequences among all species used in this study, see details in Appendix 2. The morphological measurements of these specimens were displayed in Table 1, morphological characteristics include tympanum absent, dorsal surface of head and body smooth, within small and sparse granules, heels not meeting when flexed hind-limbs to body axis, and undeveloped toe pads, which are largely consistent with B. cryptotympanicus (Fig. 2). Further phylogenetic analysis revealed that the specimens clustered within a branch of B. cryptotympanicus. Based on the uncorrected distance model of the 16S rRNA gene, the average genetic distance within B. cryptotympanicus is 0.2%, significantly lower than the genetic distances observed among species within the genus Bufo, which averaged 4.35% (Appendix 2).Combining morphological characteristics with phylogenetic analysis confirms that the newly collected specimens belong to B. cryptotympanicus of the family Bufonidae,making a new amphibian record for Hunan Province, China.