The European Union’s Bioeconomy Strategy aims to the transition to a circular, sustainable bio-based economy. It should be highlighted that bio-based production systems face significant challenges concerning the sustainable availability of the biological materials that are used as feedstocks for bio-based products. The development of sustainable value chains of biological feedstocks is a challenging prerequisite for the implementation of the Bioeconomy Strategy and Green Deal priorities. An important step towards this goal is to develop a methodological approach for mapping the current European biological feedstocks by identifying their source, trade, and fate. In the material to follow, a general, comprehensive methodology for carrying out a Material Flow Analysis (MFA) of the main biological feedstocks that can be used by the bio-based industries is presented. The MFA methodology is implemented for the main agro-industrial waste and by-product streams in the Region of Central Macedonia (RCM) in Greece, identifying the most promising locally available biological feedstocks. Approximately 1.4 million tons (dry matter) residual biomass per year are harvested in the RCM mainly from cereals and industrial crops. The majority of this biological feedstock is valorized in conventional uses for bioenergy and for feed. Constrains regarding their use (e.g. legal, social, technical etc.) are also discussed. The outcomes of this study are expected to contribute to the development of the local circular bio-based economy and to act as a paradigm for other national (Greek) and European regions.