Crop-to-crop transgene flow may affect the seed purity of non-transgenic rice varieties, resulting in unwanted biosafety consequences. The feasibility of a rapid and nondestructive determination of transgenic rice seeds from its non-transgenic counterparts was examined by using multispectral imaging system combined with chemometric data analysis. Principal component analysis (PCA), partial least squares discriminant analysis (PLSDA), least squares-support vector machines (LS-SVM), and PCA-back propagation neural network (PCA-BPNN) methods were applied to classify rice seeds according to their genetic origins. The results demonstrated that clear differences between non-transgenic and transgenic rice seeds could be easily visualized with the nondestructive determination method developed through this study and an excellent classification (up to 100% with LS-SVM model) can be achieved. It is concluded that multispectral imaging together with chemometric data analysis is a promising technique to identify transgenic rice seeds with high efficiency, providing bright prospects for future applications. © 2013 Elsevier Ltd. All rights reserved.