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Background: Currently in Australia, men are deferred from donating blood if they have had sex with another man within the past 3 months. However, a proposed gender‐neutral assessment (GNA) process will ask all donors questions about sex with new or multiple recent partners, with deferral based on responses to a question about anal sex. Understanding the acceptability of such questions among existing and potential blood donors is paramount for successful implementation of GNA. Study Design and Methods: We used data from a nationally representative survey to estimate the levels of comfort with the proposed GNA questions among the Australian population and subgroups, defined by self‐reported ethnicity and religion. Respondents were aged over 18 and living in Australia. Results were weighted to represent the population. Results: Most of the 5178 respondents described themselves as comfortable with answering questions about new partners (73.1%) or anal sex (64.0%) to donate blood. However, 2.2% and 4.5% indicated that questions about new sex partners and anal sex, respectively, would stop them from donating, and 4.4% and 7.7% respectively, said they were "completely uncomfortable." By religion, the least comfortable were Muslim or Eastern Orthodox respondents, and by country of birth, the least comfortable were those born in the Middle East, followed by those born in Southern Europe and Asia. Discussion: GNA appears to be broadly acceptable in the Australian context, but our findings suggest that key GNA questions are less acceptable in some population subgroups, indicating a need for targeted campaigns that consider cultural sensitivities. [ABSTRACT FROM AUTHOR]