Abstract:Bufo rubroventromaculatus is a newly discovered species of the genus Bufo from northern and central Vietnam in 2024. Currently, its distribution in China has been recorded only in Napo County, Guangxi Zhuang Autonomous Region and Malipo County, Yunnan Province—areas along the border between China and Vietnam. Its presence in other regions of China remains uncertain. Since 2022, eight specimens of Bufo species have been collected from Dawuling Forestry Station, Xinyi City, Guangdong and three from Guangxi Yachang Orchid National Nature Reserve, Leye County, Guangxi (Fig. 1). The morphological characteristics of these specimens were examined and measured via a digital vernier caliper for preliminary species identification. To further confirm species delimitation, we used MEGA 11, raxmlGUI 1.3, and MrBayes 3.2.4 to construct phylogenetic trees. In addition, uncorrected p-distances were calculated in MEGA 11 based on mitochondrial 16S rRNA, COI, and ND2 gene sequences. Morphological comparisons revealed that these specimens matched the diagnostic characteristics of B. rubroventromaculatus (Table 2 and Fig. 2). These specimens had the following differences from the type specimens:head width larger than length; vomerine teeth present or absent; tympanum prominent; dorsal skin warts smooth with relatively few keratinized spines; snout-vent length larger than the type series in males. The phylogenetic analysis based on mitochondrial 16S rRNA, COI, and ND2 genes showed that all specimens of Bufo species clustered into a monophyletic lineage with a highly support rate (Fig. 1). The genetic distances between the populationfrom Xinyi City, Guangdong and the population fromLeye County, Guangxi, China were 0.36% (16S rRNA), 2.48% (COI), and 2.33% (ND2), respectively, while the genetic distance between these two populations and the Vietnamese population were 2.46% (Guangdong) and 0.86% (Guangxi) based on the ND2 gene, respectively (Appendices 1﹣3). Taking into account the results of morphological comparison and phylogenetic analysis, we confirmed that the collected specimens belonged to B. rubroventromaculatus. This represents a new record of this amphibian species in Guangdong. The discovery expands the known distribution of B. rubroventromaculatus from southeastern Yunnan and western Guangxi to northwestern Guangxi and western Guangdong. It provides significant insights into the phylogeography and biogeography of the genus Bufo. Moreover, this study refines the morphological description and biological information of B. rubroventromaculatus.