Exploring the driving forces promoting speciation is a key question in evolutionary biology. Cycas bifida and C. micholitzii are two leaflets dichotomously divided Cycas species that have morphological similarities. Their distribution areas are separated by the RRFZ, a biogeographical barrier for Cycas species. The hypothesis for explaining their divergence is geographical isolation or convergent evolution. Three chloroplast DNA fragments, five low copy nuclear genes and 16 microsatellite loci were used to test this hypothesis and also to reveal their population genetics. The two species had high genetic differentiation but low gene flow with a deep divergence occurred in the late Miocene. The long-term geographical isolation not convergent evolution could explain for the divergence of the two species revealed from significant IBD testing, Barrier analysis and Niche consistency detection. Subsequently, each species made its own response to the Pleistocene climate fluctuations: a weak bottleneck effect in C. bifida and a population expansion in C. micholitzii. These findings imply that the role of geographic isolation rather than convergent evolution facilitates them divergence by constraining gene flow. Maybe they have gene flow or introgression with their neighboring distribution of Cycas species, making them genetically closer to neighboring Cycas species.