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The productivity, distribution and population structure of poplar are affected by temperature transitions. Poplar floral buds develop in a fluctuating environment and the molecular basis of temperature-dependent flowering regulation has been extensively studied, but little is known about how sex-specific floral bud development responds to temperature transitions. Here, morphological observations indicated that floral bud growth rates were affected by maximum and minimum air temperature at the later stages of enlargement (stage 4) and later stage of dormancy (stage 8), respectively. We investigated the physiological, biochemical and gene expression changes in floral development and in response to temperature treatment (heat and chilling stress). Male floral buds showed more adverse effects than female floral buds under temperature treatment. Temperature treatment experiments revealed that temperature treatment significantly increased catalase, peroxidase, superoxide dismutase activities and transcription of related genes in female floral buds, whereas malondialdehyde (MDA) significantly increased only in males. Soluble sugars and protein increased both in female and male floral buds but were higher in males. Temperature treatment also caused significant increases in Ca2+ content and transcription of genes related to calcium transport in female flowers. These results revealed sex-specific floral developmental responses to seasonal temperature transitions and suggest that in Populus tomentosa, female floral buds possess better mechanisms for environment adaptation than do males.