Ecological adaptation to environmental changes is a strong driver of evolution, enabling speciation of pelagic plankton in the open ocean without the presence of effective physical barriers to gene flow. The tropical ocean environment, which plays an important role in shaping marine biodiversity, has drastically and frequently changed since the Pliocene. Nevertheless, the evolutionary history of tropical pelagic plankton has been poorly understood, as phylogeographic investigations are still in the developing state and paleontological approaches are insufficient to obtain a sequential record from the deep-sea sediments. The planktonic foraminifer Pulleniatina obliquiloculata is widely distributed in the tropical area throughout the world's oceans, and its phylogeography is well established. It is thus one of the best candidates to examine how past environmental changes may have shifted the spatial distribution and affected the diversification of tropical pelagic plankton. Such an examination requires the divergence history of the planktonic foraminifers, yet the gene marker (partial small subunit (SSU) rDNA) previously used for phylogeographic studies was not powerful enough to achieve a high accuracy in estimating the divergence times. The present study focuses on improving the precision of divergence time estimates for the splits between sibling species (genetic types) of planktonic foraminifers by increasing the number of genes as well as the number of nucleotide bases used for molecular clock estimates. We have amplified the entire coding regions of two ribosomal RNA genes (SSU rDNA and large subunit (LSU) rDNA) of three genetic types of P. obliquiloculata and two closely related species for the first time and applied them to the Bayesian relaxed clock method. The comparison of the credible intervals of the four datasets consisting either of sequences of the partial SSU rDNA, the complete SSU rDNA, LSU rDNA, or a combination of both genes (SSU+LSU) clearly demonstrated that the two-gene dataset improved the accuracy of divergence time estimates. The P. obliquiloculata lineage diverged twice, first at the end of the Pliocene (3.1 Ma) and again in the middle Pleistocene (1.4 Ma). Both timings coincided with the environmental changes, which indirectly involved geographic separation of populations. The habitat of P. obliquiloculata was expanded toward the higher latitudinal zones during the stable warm periods and subsequently placed on the steep environmental gradients following the global cooling. Different environmental conditions in the stable warm tropics and unstable higher latitudes may have triggered ecological divergence among the populations, leading to adaptive differentiation and eventually speciation. A comprehensive analysis of divergence time estimates combined with phylogeography enabled us to reveal the evolutionary history of the pelagic plankton and to find the potential paleoenvironmental events, which could have changed their biogeography and ecology.