OBJECTIVE: To systematically review health care professionals' practices and attitudes toward addressing sexuality with people who are living with chronic disease and disability. DATA SOURCES: Scopus, PubMed, PsycINFO, Cumulative Index to Nursing and Allied Health, Allied and Complementary Medicine Database, and MEDLINE were searched to August 2020 for English language publications. Reference lists of relevant publications were also searched. STUDY SELECTION: Eligible studies reported on knowledge, attitudes, and behaviors of health care professionals about addressing sexuality in the context of chronic disease and disability. The search yielded 2492 records; 187 full texts were assessed for eligibility and 114 documents were included (103 unique studies). Study quality was rated using the Mixed Methods Appraisal Tool. DATA EXTRACTION: Characteristics of included studies were recorded independently by 2 authors. Differences were resolved through discussion or by a third author. DATA SYNTHESIS: A sequential, exploratory mixed studies approach was used for synthesis. Pooled analysis showed that 14.2% (95% CI, 10.6-18.9 [I2=94.8%, P<.001]) of health professionals report routinely asking questions or providing information about sexuality. Professionals reported limited confidence, competence, and/or comfort when initiating conversations about sexuality or responding to patient questions. Sexual rehabilitation typically focused on the effect of disease, disability, and medication on sexual function. Broader dimensions of sexuality were rarely addressed. CONCLUSION: Despite recognizing the value of sexuality to health and well-being, most health professionals regardless of clinical context fail to routinely include assessment of sexuality in their practice. Professionals have limited knowledge and confidence when addressing sexuality and experience significant discomfort when raising this topic with people living with chronic disease and disability. Multicomponent implementation programs are needed to improve health professionals' knowledge, competence, and comfort when addressing sexuality for people living with chronic disease and disability.