【Objective】This study aimed to investigate the harm of low temperature in the cotton (Gossypium hirsutum L.) seedling stage on floral bud differentiation and the effect on seedcotton yield, to analyze the change characteristics of cotton floral bud differentiation phenotypes and terminal buds endogenous hormones under low temperature, so as to provide the theoretical basis for the high-quality and high-efficiency cultivation technology of cotton under low temperature.【Method】Using the early-maturing and high-quality cotton variety Zhong 425 as the material, a pot experiment was conducted in the smart greenhouse of the Pailou Experimental Station of Nanjing Agricultural University from 2022 to 2023 to simulate the daily average temperature environment during the cotton seedling stage in Aksu, southern Xinjiang. Two temperature treatments were set up: the control (CK, with a daily average temperature of 27 ℃, and daily maximum and minimum temperatures of 32 and 22 ℃, respectively) and the low-temperature treatment (LT, with a daily average temperature of 20 ℃, and daily maximum and minimum temperatures of 25 and 15 ℃, respectively). The number, size, and morphological anatomical structure of cotton flower bud differentiation were investigated, and the changes in endogenous hormones in shoot apices under low temperature during the seedling stage were analyzed. Additionally, the changes in cotton bolls and their component biomass, as well as relevant indicators of seed cotton yield, were examined after the removal of low temperature stress during the seedling stage.【Result】During the differentiation of cotton flower buds, the increase in Indole-3-acetic acid (IAA) content and the decrease in trans-Zeatin-riboside/Gibberellin A3 (ZR/GA3 ) ratio in the terminal buds of cotton under low temperature during the seedling stage inhibited flower bud differentiation. Meanwhile, the content of abscisic acid Abscisic Acid (ABA), GA3, and ZR increased in response to the adverse effects of low temperature. Changes in endogenous hormones in the shoot tips caused by low temperature during the seedling stage slowed down the process of flower bud differentiation. When the flower buds of the first fruit node on the first fruit branch differentiate from the bract differentiation stage to the sepal differentiation stage, petal-stamen differentiation stage, pistil differentiation stage, and sexual organ formation stage, the leaf age increased by 16.6%-19.4%, 26.5%-31.3%, 17.6%-29.0%, 16.6%-23.3%, and 26.6%-30.0%, respectively; the number of flower buds at the 4-leaf-1-heart, 5-leaf-1-heart, and 6-leaf-1-heart stages of cotton seedlings decreases by 33.3%-55.2%, 24.0%-53.1%, and 26.8%-32.9%, respectively. Due to the slow growth and development of cotton seedlings under low temperature during the seedling stage, the number of flower buds in cotton seedlings exposed to the same number of days of temperature treatment decreased more significantly, with reductions of 66.7%-85.7%, 74.0%-87.8%, and 70.7%-81.7% compared with the control group at the 4-leaf-1-heart, 5-leaf-1-heart, and 6-leaf-1-heart stages, respectively; the sizes of flower buds at these stages also decreased by 33.3%-36.4%, 70.7%-71.6%, and 44.6%-48.3%, respectively. After the removal of low temperature stress during the seedling stage, the development of cotton bolls was still affected, with significant reductions in boll and its component biomass. Specifically, the biomasses of boll shell, fiber, and cottonseed decreased by 64.6%, 65.5%, and 66.7%, respectively. The number of cotton bolls decreased by 65.4%, ultimately leading to a 65.5% reduction in seed cotton yield.【Conclusion】Under low temperature conditions during the seedling stage, the increased IAA content and decreased ZR/GA3 ratio in the apical buds of cotton inhibited the differentiation of pre-summer peach flower buds. Low temperature during the seedling stage retarded the reproductive development of cotton by delaying flower bud differentiation, which reduced the biomass of cotton bolls. Low temperature at this stage also decreased the number of flower buds, ultimately leading to a reduction in the number of cotton bolls and lower seed cotton yield.