The objective of this study was to find out the influence of altitude of coffee plant and soil pH and organic carbon on alkaloid contents of green coffee beans. The alkaloids content of 54 green coffee beans samples collected from coffee plants in Sidama, Illubabor, Jimma, Wellega and Gedeo in southern region of Ethiopia grown at different altitudes (1515-2220 masl)was determined by high performance liquid chromatography. The caffeine and trigonelline were found in the range of 0.68-1.74% (w/w) and 0.68-1.44% (w/w), respectively. Theobromine was detected in only 18 samples and ranged 0.0186-0.32% (w/w). It was not detected in 36 samples. Theophylline was not detected in any of the green coffee beans samples. A weak negative correlation (R = -0.222) was found between the caffeine contents and the altitude of the coffee plants while a very weak positive correlation (R = 0.072) was found between the trigonelline contents and the altitude of the coffee plants. A strong negative correlation (R = -0.775) was found between the trigonelline contents and the caffeine content. A weak negative correlation was found between the caffeine contents and the soil organic carbon (R = -0.279) and between the trigonelline contents and the soil organic carbon (R = -0.101) while a weak positive correlation was found between the caffeine contents and the soil pH (R = 0.173) and between the trigonelline contents and the soil pH (R = 0.358) at which the coffee plants were grown.