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Objectives: Many factors such as personal and cultural beliefs, misinformation, fear of death and inadequate will registering procedures can influence post‐mortem organ donation. The present study aimed to explore the perceptions, beliefs and information around post‐mortem donation and will expression in different groups of the Italian population, to orient future interventions and raise awareness. Design: Qualitative research with focus groups. Methods: A total of 38 focus groups involving 353 participants including the general population (young adults: 18–39, mature adults: 40–70), local and hospital health professionals, critical area health professionals (emergency room and intensive care), registry office employees and opinion leaders, were conducted in six regions from different parts of Italy between June and November 2021. Thematic analysis was conducted with the use of Atlas.ti9. Results: Five overarching themes were identified: dilemmas regarding donation, resistance to donation, facilitators of donation, difficulties in terms of will expression and proposals to encourage will expression. Possible facilitators were having personal and professional experiences with organ donation, feeling useful for society, having reliable information and trust in the health care system. Potential barriers to donation were doubts and fears about brain death, bodily integrity concerns, religious beliefs, misinformation and distrust in the health care system. Conclusions: These results highlighted the significance of a bottom‐up perspective with regard to identifying the personal perceptions and beliefs with regard to donation, underlining the urgency of creating tailored interventions to sensitize different groups of the population in terms of promoting an informed choice and a culture of donation. [ABSTRACT FROM AUTHOR]