Circumscriptions for the Sapindaceae family and its infrafamilial relationships have been widely discussed. Certain groups are highly morphologically similar; thus, it is difficult to identify certain taxa. DNA Analyses have also indicated complex phylogenetic relationships, and it is difficult to relate such analyses to morphological data. Given the above concerns, this study aimed to investigate the fatty-acid profiles of the seed oils of 16 Sapindaceae species belonging to five tribes and to evaluate their potential chemotaxonomic significance. In total, eleven fatty acids were identified, and eicosenoic acid predominated in nine species. Multivariate analyses (principal component and cluster analyses) of the fatty-acid profiles of the seed oils allowed to separate them in two major clusters. The first cluster, characterized by oils with high eicosenoic acid levels, included all species belonging to the Paullinieae tribe (Cardiospermum, Paullinia, and Serjania species). In the second main cluster, the chemical similarity of the oils was lower, and the species belonged to different tribes. Nevertheless, the tree investigated Allophylus species (Thouinieae tribe) constituted a separate subcluster. Thus, the results showed that the fatty-acid composition of the seed oils of Sapindaceae species provide chemotaxonomic support for the separation of the Paullinieae tribe from the other tribes studied.