Objective This study investigated teachers’ experiences with LGBTQ+ instruction as well as other school environment changes related to LGBTQ+ issues. It looked at teachers’ attitudes towards LGBTQ+ individuals, comfort levels with the topics and administrative support, self-perceived knowledge and teacher preparation, and supports and barriers to instruction. Lastly, the study investigated teachers’ in-service and pre-service training experiences. Design Using a sequential mixed methods approach, research began with qualitative inquiry, followed by quantitative assessment via self-administered paper surveys. Methods There were 11 in-depth participant interviews, and 94 surveys assessing teachers’ attitudes, comfort levels, self-perceived knowledge and preparation, and supports and barriers to LGBTQ+ topics. Results Data analysis revealed teachers’ school location and pre-service and in-service training experiences impacted comfort levels and self-perceived knowledge and preparation levels to teach these issues. Data also indicated that new teachers were the least comfortable with LGBTQ+ topics in the classroom and school environment. Conclusion Data demonstrated lack of LGBTQ+ teacher training, indicated need to support new Health Education professionals, and inadequate pre-service teacher training.