Abstract The interdigital webs of aquatic tetrapods are a key structure evolved for adaptation, which are formed by inhibiting interdigital cell death (ICD). Diverse interdigital morphologies have independently evolved among species, and the regulatory mechanisms responsible for their development are still not fully understood. The Chinese soft-shell turtle (Pelodiscus sinensis) serves as a good research model that exhibits transitional traits from webless to fully webbed. In this study, we collected eight samples of interdigital webs from the fore- and hindlimbs of turtles at embryonic stage 19 (TK19) for RNA sequencing (RNA-seq) analysis. We identified 608 differentially expressed genes (DEGs). Whole-mount in situ hybridization (WISH) and real-time quantitative PCR (RT-qPCR) of representative genes confirmed the accuracy of the transcriptomic results. These findings not only provide new perspectives and data to support comparative studies of adaptive convergent evolution in aquatic animals but also enhance our understanding of the mechanisms underlying tetrapod limb morphogenesis. Furthermore, these results provide potential molecular targets for research on the plasticity of programmed cell death or senescence.