In the APG III, the family Berberidaceae is placed in the Ranunculales within the basal Eudicots. Here, the phylogenetic relationships within Berberidaceae were reconstructed using the Maximum Likelihood method on sequence data from four genomic regions (nuclear ribosomal 26S rDNA and plastid rbcL, matK, and trnLF). Based on previously reported phylogenetic tree and pollen observation, the pollen morphologies of 16 genera of this family were investigated. Pollen plesiomorphy，synapomorphy, and evolutionary pattern were revealed by Fitch Parsimony analysis. Ten pollen characters, including dispersal unit, polarity, shape class, size, aperture number, aperture position, ectoaperture shape, supratectal elements, tectum sculpture, and exine thickness, were evaluated to reassess phylogenetic relationships. Tracing pollen character evolution showed that Berberidaceae possessed a series of plesiomorphies, such as the monad pollen unit, isopolarity, spheroidal shape and medium size. Apolarity, multiple apertures and global aperture state were found to be synapomorphies for the Berberidoideae, strongly supporting the monophyly of this subfamily. Triaperture was a synapomorphy for both Podophylloideae and Nandinoideae, respectively. Berberidoideae and Nandinoideae shared the synapomorphy supratectal elements absent, which distinguished them from Podophylloideae, and supported the sister relationship between Berberidoideae and Nandinoideae. Pollen evolutionary significance of some genera was discussed, and several pollen characters were suggested to be used for defining certain genera, such as Bongardia and Ranzania.