Rhinogobius zhoui is found only in streams on Lianhua Mountain, Guangdong Province, China. Juveniles of Rhinogobius zhoui bred in an artificial environment do not undergo a planktonic period and have a landlocked life history. At present, there is not much published literature regarding this species. To study the structural characteristics and phylogenetic information of the mtDNA of Rhinogobius zhoui, the mtDNA of R. zhoui was obtained by next-generation sequencing. The mtDNA was assembled, annotated, and analyzed using a bioinformatics method, followed by alignment of mtDNA and analysis of sequence differences. R. zhoui was analyzed phylogenetically using data on 48 species of Gobiiformes and 2 species of Cypriniformes downloaded from NCBI ( https://www.ncbi.nlm.nih.gov/ ), and phylogenetic trees were generated using maximum likelihood and Bayesian inference. The results of our analysis showed that (1) the length of the mtDNA of R. zhoui was 16,491 bp, according to next-generation sequencing and its mitochondrial structure included 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one non-coding region and (2) from the phylogenetic tree thus generated, it can be seen that R. zhoui and Rhinogobius duospilus are closely related. This study will re-examine the Phylogenetic tree of the major lineages in Gobiiformes.