Fossil leaves of Populus L. (Salicaceae Mirb.) were widely reported in the North Hemisphere during the Cenozoic. However, most of them are leaf impressions without details of cuticles or only preserved as tiny cuticle fragments. Here, we describe several fossil leaves of Populus with fine cuticular structures from the upper Pliocene of Tengchong, western Yunnan, China. The leaves have ovate, rhombic-ovate to orbicular leaf shape with long petioles, pinnate venation, rounded to cuneate bases, acuminate apices and serrate margins. They are pilose bilaterally with multicellular trichome bases and hypostomatic with paracytic stomata. According to the gross morphology and cuticle structures, the present fossils mostly resemble the extant species P. rotundifolia Griff. except that the fossils have denser trichome bases, and are therefore tentatively assigned to Populus cf. rotundifolia Griff. Nowadays, P. rotundifolia is distributed at mountain slopes above an elevation of 2000 m a.s.l. The present new finding indicates that western Yunnan might have approached its present height at least by the late Pliocene. Recent phylogenetic study proposed that the divergence of P. rotundifolia from P. davidiana Dode occurred at the Pleistocene (Calabrian, ca. 0.88 Ma). However, our fossil occurrence implies that such a divergence might have occurred at least by the late Pliocene (3.3–2.8 Ma), much earlier than the molecular estimation. In addition, the Hengduan Mountainous Region might be the species differentiation center of P. rotundifolia from P. davidiana based on their modern distributions and the present fossil occurrence.