Abstract: To explore the effect of drought to stomatal development in woody plants, five Populus deltoides× P. nigra clones, i.e., R270 (P. deltoides× P. nigra), NE-19 P. nigra× (P. deltoides× P. nigra), 107 (P.× euramericana ‘74/76'), 109 (P. deltoides× P. alba ‘Mincio') and 111 (P. deltoides× euramericana ‘Bellotto') were selected to undergo 14 days of drought and 7 days of re-watering treatment. The results showed that photosynthesis of NE-19, R270 and 107 returned to the normal level at the fifth day after re-watered, with stomatal conductance back to normal level after 3 days. However, 109 and 111 recovered slowly. As for the growth rate after re-watered, the order from high to low was NE-19 (64.96%), R270 (55.73%), 107(49.87%), 109(35.08%), and 111(23.62%). According the results of photosynthesis, stomatal conductance and growth rate, the drought tolerance was ranked as NE-19 > R270 > 107 > 109 > 111. For all five clones, the stomatal development slowed down when subjected to the drought, indicated by the stomatal index (SI) in young leaves. The SI of NE-19 and R270 had the most remarkable change during the drought, being 30.75% and 29.24%, respectively. The clones with better drought tolerance may be more positively responsive to ambient water content change through stomatal development. Using qRT-PCR we tested the difference of transcript abundance of five genes associated with stomatal development between control and drought treatment groups. EPFL9 and FAMA, which positively control stomatal development, were down-regulated during drought, but ERECTA and EPF1 which negatively regulate the process were up-regulated during drought, and SDD1 had no change during the whole treatment. Our findings highlight that poplar inhibits stomatal development through adjusting expression of ERECTA, EPF1, EPFL9 and FAMA in immature leaves to reduce water loss so as to withstand drought stress.