The comparative pollination biology of a population of Primula beesiana, a population of P. bulleyana and an overlapping population of these two species in the Yulong Shan, Yunnan Province, China, was studied in 2004 and 2005. The results indicated that both P. beesiana and P. bulleyana were typical heterostylous and obligate outcrossing species; the main pollinators of the two species were bees and butterflies. In all three populations’ sites, the main pollinating visitors of the two species showed preference to one of the two species, resulting in pollinators’ ethological isolation of the two species. The pollinators’ ethological isolation contributes to the reproductive isolation of the two species, which supports the hypothesis that P. beesiana and P. bulleyana are two distinct species. The reproductive isolation of the two species (ethological isolation) is probably an important mechanism in maintaining species boundaries in the genus, and has contributed to the species diversification of Primula in the area. In addition, gene exchanges between P. beesiana and P. bulleyana occurred to some extent in the overlapping population, but whether natural hybridization has contributed to species diversification in Primula remains to be seen.