The effects of different ecological conditions on morphological and physiological features in squaring stage of tobacco cultivar K326 (Nicotiana tabacum L cv. K326) were investigated under different field conditions. The test fields with different ecological conditions were located at Sijie (S), Dayingjie (D) and Xiaojie (X) in Yuxi City of Yunnan province, respectively. The tobacco planted in D showed best morphological characters. However, the tobacco in S showed worst morphological characters. Flavonoid content, chlorophyll content and ratio of chlorophyll a/b had no significant difference among the three planting sites. Content both of carotenoid and soluble protein was the highest, while ratio of chlorophyll/carotenoid was the lowest in tobacco at S. The order of P_nmax, LCP and LSP was test S>test D>test X. When PAR ranging from 800 μmol·m^-2·s^-1 to 1000 μmol·m^-2·s^-1, the order of C_i, G_s and T_r was test D>test X>test S, and the order of WUE was test S>test X>test D. In addition, to ascertain the effects of ecological factors on morphological and physiological features of tobacco in different sites, the values of gray association degrees were calculated. The results show that the main ecological factors which influencing stem length, leaf area and contents of flavonoid, MDA and chlorophyll in K326 were light intensity, temperature, precipitation, UV-B radiation and illumination time, while content of alkali-hydrolyzable nitrogen in planted soil was regarded as the major influencing factor on carotenoid soluble protein and rate of P_nmax in K326.