Premise of the study: Cycas is an important gymnosperm with the most diverse of all cycad genera. The taxa within Cycas taiwaniana complex are morphologically similar and difficult to be distinguished by a lack of genomic resources. Methods: We characterized transcriptomes of two closely related and endangered Cycas species endemic to Hainan, China: C. hainanensis and C. changjiangensis. Furthermore, we sequenced three single copy nuclear genes for the Cycas taiwaniana complex developed from transcriptome. Then we evaluated species boundaries based on the multispecies coalescent method implemented in BPP. Results: We obtained 68,184 and 81,561 unigenes for C. changjiangensis and C. hainanensis, respectively. The estimated divergence time revealed the two related species diverged more recently. Six positively selected genes are mainly involved in stimulus responses, suggesting that environmental adaptation may play an important role in the divergence of the two species. The similar peak at 1.0 of Ks distributions for paralogs indicated a common whole-genome duplication event. Results of species delimitation indicated the Cycas taiwaniana complex consisted of three distinct lineages, which corresponds to morphological differentiation. Discussion: Our study provides evidence from transcriptome for taxonomical treatment of the Cycas taiwaniana complex and new insights into evolution of this living fossil genus.