Bioeconomy is no longer limited to being a concept in research papers and policy strategies; it presents real opportunities for regional development, i.e., by improving local employment, socio-economic development, economy and sustainable local bioresource use. But the obtaining of these opportunities is challenged by various barriers. It is proposed that analogously with other widely research fields (energy efficiency, industrial sustainability), the implementation of novel bioeconomy ideas, even when they are scientifically based, encounters the so-called ‘implementation gap’ (similarly to, e.g., the ‘energy efficiency gap’). This implementation gap is related to national, but also regional challenges, i.e., technical, economic, organizational, behavior and other barriers that hinder the most successful bioeconomy deployment. The technical obstacles can be, e.g., lack of processing or production technologies, capacity problems, while organizational barriers can manifest as logistics chain problems due to the availability of bioresources or internal organization problems of the company, lack of time, reluctance to take on new responsibilities or lack of awareness of opportunities provided by the bioeconomy. This significance of the regional analysis level for barrier assessment calls for more precise information. The first step for in-depth investigation of the bioeconomy implementation gap in Latvian context is the research of the bioeconomy ecosystem. In fact, as required by the National industrial policy guidelines, the Strategy for knowledge based bioeconomy development for Latvia was developed in 2022 by the Latvian bioeconomy ecosystem stakeholders. The current research focuses on the identification and more detailed characterization of the stakeholders of Latvian bioeconomy ecosystem, their interactions, as well as the identification of current challenges and barriers for knowledge-based bioeconomy and innovations development. In order to further elaborate the structure of the bioeconomy ecosystem, the mapping of ecosystem stakeholders was implemented.