Abstract: There has been controversy about the genetic relationship between Hyriopsis cumingii and Hyriopsis cumingii. To further analyze the phylogeny of freshwater pearl mussels, we conducted comparative transcriptomic analysis between H. schlegelii and its relatives, including Hyriopsis cumingii, Anodonta woodiana and Cristaria plicata. The genetic distance between the four freshwater pearl mussels was calculated after the multiple sequence alignment (MSA) of the single-copy orthologs their shared. The results showed that the genetic distance between H. schlegelii and H. cumingii was only 0.00746 (<1%), while the genetic distance between other freshwater pearl mussels was almost 10 times bigger. Phylogenetic analysis showed that Ambleminae and Anodontinae diverged approximately 51.44 million years ago (Mya), while H. schlegelii and H. cumingii were divided about 4.24 million years ago. Further analysis identified the specific genes in the transcriptome of H. schlegelii. The enrichment of the KEGG pathway indicated that these genes were mainly enriched in pathways such as CD molecules and protein digestion and absorption. Moreover, GO annotation results showed that there were many specific genes related to H. schlegelii excellent characteristics, including development-related biological processes, such as developmental process, system development, animal organ development, and immune-related biological process, immune system process and antigen processing and presentation in GO classification. The above results showed that H. schlegelii and H. cumingii are closely related, and may be different subspecies or populations, which has important reference significance for the germplasm conservation and genetic breeding of freshwater pearl mussels. In addition, compared with H. cumingii, H. schlegelii may have several specific genes related to development and immunity, which provides some clues for exploring the related molecular mechanism.