Tempe, a fermented soybean originally from Indonesia, is an excellent protein source with high nutritional quality. Differences in the production process and unique fermentation condition in different regions result in varieties of tempe. Despite its high cultural and economic values, there are very few studies on the characterization of tempe based on the differences of production process and geographical origin. Metabolomics is a powerful tool assessing food quality, food safety, and determination of origin and varietal differences. In this study, metabolomics is applied for the study of Indonesian tempe obtained from various regions and different production processes. Seventeen samples were collected from 6 different cities in Java Island, which were produced by local tempe crafters (traditional), semi-modern industry and modern industry. Untargeted metabolomics by gas chromatography coupled with mass spectrometry (GC/MS) was implemented to discriminate various kinds of tempe and identify metabolites that are associated with these differences. Results showed that tempe produced in different places clustered together according to the cities and their production category. Sugars and amino acids groups were found to be primary compounds that contributed to this result. This is the first report that address the metabolic differences between different varieties of tempe from different regions and production processes. The knowledge from this study is important for future development of tempe production. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.