ObjectiveTo examine vaccine uptake, hesitancy and explanatory factors amongst people with serious and/or chronic health conditions, including the impact of underlying disease on attitudes to vaccination.DesignCross-sectional survey.SettingTen Australian health services.Participants4683 patients (3560 cancer, 842 diabetes and 281 multiple sclerosis) receiving care at the health services participated in the 42-item survey, between June 30 to October 5, 2021.Main outcome measuresSociodemographic and disease-related characteristics, COVID-19 vaccine uptake, and the scores of three validated scales which measured vaccine hesitancy and vaccine-related beliefs generally and specific to the participants’ disease, including the Oxford COVID-19 Vaccine Hesitancy Scale, the Oxford COVID-19 Vaccine Confidence and Complacency Scale and the Disease Influenced Vaccine Acceptance Scale. Multivariable logistic regression was used to determine the associations between scale scores and vaccine uptake.ResultsOf all participants, 81.5% reported having at least one COVID-19 vaccine. Unvaccinated status was associated with younger age, female sex, lower education and income, English as a second language, and residence in regional areas (all p<0.05). Unvaccinated participants were more likely to report greater vaccine hesitancy and more negative perceptions toward vaccines (all p<0.05). Disease-related vaccine concerns were associated with unvaccinated status and hesitancy, including greater complacency about COVID-19 infection, and concerns relating to vaccine efficacy and impact on their disease and/or treatment (all p<0.05).ConclusionsDisease-specific concerns impact COVID-19 vaccine-related behaviours and beliefs in people with serious and/or chronic health conditions. This highlights the need to develop targeted strategies and education about COVID-19 vaccination to support medically vulnerable populations and health professionals.Trial registrationACTRN12621001467820