Variability in oil content and seed weight of 37 accessions of mahua (Madhuca longifolia Koenig) J.F. Macribide collected from different part of Tamil Nadu, India were assessed. There were significant differences in 100-seed weight and oil parameters, namely, kernel oil %, palmitic acid, stearic acid, oleic acid and linoleic acid. Maximum seed weight (340 g) was recorded in IC554535 and the least weight (100 g) was recorded in IC554545. Kernel oil ranged from minimum of 44.4% in IC554535 to maximum 61.5% in IC556617. The saturated fatty acid i.e palmitic acid and stearic acid ranged from 11.7-25.9% to 19.1-32.2%, respectively. The oleic acid ranged from 32.9 to 48.7% of the total fatty acid while linoleic acid ranged from 9.4 to 15.4%. High heritability was recorded for all the traits studied. It was maximum for palmitic acid (98.6%) and minimum for linoleic acid (95.8%). The Euclidean pairwise dissimilarities were calculated and clustered by UPGMA based SAHN clustering method. Maximum and minimum Euclidean pairwise dissimilarities observed were 7.01 and 0.46, respectively. All the 37 accessions were grouped into three major clusters. The accessions in cluster II had low palmitic acid and high oleic acid while cluster III had high palmitic acid and low oleic acid content. Normalized Mantel statistics (r = 0.8) and principal component analysis supported cluster analysis. Thus on the basis of present findings it is suggested that hybridization between accessions of cluster II and III will result in wide spectrum of variability in subsequent generations for medicinal, edible applications and biodiesel production. (C) 2011 Elsevier Ltd. All rights reserved.