A good knowledge about Science, Technology, Engineering, and Mathematics (STEM) integration is essential for chemistry teachers before they are implementing a successful chemistry learning using STEM approach. This paper reported in-service chemistry teachers' prior knowledge in integrating STEM in high school chemistry learning. A descriptive study was adopted in this research. A number of 82 in-service chemistry teachers who teach in senior high schools in Yogyakarta were established as the research sample. Research sample was drawn from Sleman and Bantul Regency, Yogyakarta, Indonesia through saturated sampling. The initial knowledge about STEM of in-service chemistry teachers were captured using STEM Knowledge Questionnaire (STEM-KQ) consisting of 7 open-ended questions. The in-service chemistry teachers' response toward this STEM-KQ were analysed following qualitative descriptive method using content analysis with interpretative coding. The findings of this study revealed that in-service chemistry teachers have understand the basic concept of STEM, still it needs to be enhanced. Most of in-service chemistry teachers have known the definition and position of STEM. However, they still have a lack knowledge about the example of STEM implementation in the chemistry lesson. Therefore, the in-service chemistry teachers need to follow workshop that provide guidance on how to implement STEM approach in chemistry learning. Thus, through this activity, the in-service chemistry teachers develop their skills in preparing STEM learning that implied on their readiness in integrating STEM in the chemistry learning activities.