Background. The concept of sub-ecosystems, widely used in natural ecosystem theory, has never been introduced in software ecosystem analysis. It provides a perspective on software ecosystems that can be used to create better understanding of them and more effective ecosystem health analysis. Objectives. The objective of this research is to introduce the concept of sub-ecosystems to the field of software ecosystems. An extension on the Open Source Ecosystem Health Operationalization for measuring the health of a sub-ecosystem is created and evaluated with three small case studies. Method. A literature review of both software and natural ecosystem research is used for the definitions of key concepts. Design Science is used for the extension of the Open Source Ecosystem Health Operationalization. Finally, for the case studies, data is gathered using several data repositories and analyzed to show how the concept of sub-ecosystems is used. Results The concept of software sub-ecosystems is defined. Next to that an extension to the Open Source Ecosystem Health Operationalization (OSEHO) framework is introduced for considering sub-ecosystems in health assessments. Conclusion The subject of sub-ecosystems provides a promising new perspective on software ecosystems that improves the understanding of this research field for both researchers and practitioners. Additionally, the extended OSEHO framework can be used to more accurately measure the health of an ecosystem by looking at both larger and smaller ecosystems around it.