This study aimed to quantify and evaluate the level of Azadirachtin in neem seed kernel from ten ecotypes in Sudan over two seasons and establish relationship to the agro-ecological attributes. Extraction was done in two replicates using 12 g. After extraction, separation and concentration to 10 ml the samples quantified using HPLC. Neem trees growing in different habitats in Sudan showed variations in Azadirachtin (Az)-content depending on the climate, rainfall, soil type, latitude and altitude. The Az-content ranged from 1.08 -2.3 mg/g of the seed kernel in the first season and from 0.48 -3.09 mg/g in the second season. The average content of neem oil was 44.6% in the neem seed kernel (NSK); no variation was found between these regions in oil-content. Trees growing in regions with moderate climate, average rainfall of 400 mm, and altitude of > 470 m above sea level, proved to be rich in Az-content. However, trees growing in lower altitudes, alluvial or sandy soil, with hot climate reflected very low Az-content. Rainfall was found to be the major factor affecting the level of Az in NSK, and the optimal rainfall was 717 mm. Temperature and relative humidity had no direct effect on the Az-content. However, the combination of temperature and the rainfall showed a positive correlation with the Az level in NSKs. Latitude and altitude have direct effect on Az level. Seasonal variations were clear over the two seasons, and no relationship was found between the two seasons.