In allopatric species, the geographical isolation is generally disregarded as a legitimate cause of isolation. Beside geographical isolation, the other prezygotic isolating mechanisms in allopatric species have not been well proved to cause isolation. In order to explore if two allopatric sister species Iris dichotoma and I. domestica (Iridaceae) have other prezygotic isolating mechanisms, the natural hybrids, the positions of pollen deposited on the honeybee's body, the insect visiting period and frequency, and the daily dynamic changes of pollen quantity were detected; the daily flowering times were compared; the interspecific crossing and the pollen transfer were tested; the flower-visiting insects were categorized and identified. No natural hybrids of the two species were detected under the sympatric condition. Their daily flowering times were significantly different but had 2.6 h overlap, during which they had good cross compatibility. They had common legitimate pollinators, but the visiting time did not overlap and I. domestica’s pollen quantity was down to 0 before I. dichotoma open. The same honeybee could visit both of them as I. domestica had pollen in the net house, but their pollens were deposited on different parts of the honeybee’s body. The temporal isolation caused complete reproductive isolation, and the difference in flower structure also played an important role in reproductive isolation.