To screen out the key climatic factors determinating fat and total protein contents in cottonseeds, field experiments were conducted in the Yellow-Huai River Basin (Xuzhou and Anyang) and the lower reaches of Yangtze River Basin (Huaiyin and Nanjing) in 2005. Cotton cultivars of Kemian 1 and NuCOTN 33B were selected. Contents of fat or total protein in cottonseeds differed significantly between samples from different regions, and between flowering dates. The flowering dates, to get the highest cottonseed fat and total protein contents, were later in the Yellow-Huai River Basin than the lower reaches of Yangtze River Basin. Contents of fat and total protein significantly negatively correlated to the mean diurnal temperature difference over boll maturation period(P<0.01), and significantly positively correlated to mean daily air temperature (P<0.01), mean daily maximum air temperature(P<0.05), mean daily minimum air temperature(P<0.01) and mean daily ground temperature(P<0.01) over boll maturation period. According to the direct path analysis and stepwise regression analysis, the mean daily minimum temperature over boll maturation period was found the fundamental climatic factor. The relationship between the cottonseed fat and total protein contents and the mean daily minimum temperature well agreed with the third-polynomial regression, with determination coefficients between 0.75 and 0.9. Daily minimum temperature of 22-23 ℃ was optimum for the fat and total protein accumulation in cottonseed, and the fat accumulation would be restrained at the daily minimum temperature lower than 15℃.