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Background: Despite efforts to improve childhood immunization coverage in Nigeria, coverage has remained below the national acceptable level. In December 2019, we conducted an assessment of Missed Opportunities for Vaccination (MOV) in Ondo State, in Southwest Nigeria. The objectives were to determine the magnitude of, explore the reasons for, as well as possible solutions for reducing MOV in the State. Methods: This was a cross-sectional study using a mixed-methods approach. We purposively selected 66 health facilities in three local government authorities, with a non-probabilistic sampling of caregivers of children 0–23 months for exit interviews, and health workers for knowledge, attitudes, and practices (KAP) surveys. Data collection was complemented with focus group discussions and in-depth interviews with caregivers and health workers. The proportion of MOV among children with documented vaccination histories were determined and thematic analysis of the qualitative data was carried out. Results: 984 caregivers with children aged 0–23 months were interviewed, of which 869 were eligible for inclusion in our analysis. The prevalence of MOV was 32.8%. MOV occurred among 90.8% of children during non-vaccination visits, and 7.3% during vaccination visits. Vaccine doses recommended later in the immunization series were given in a less timely manner. Among 41.0% of health workers, they reported their vaccination knowledge was insufficient. Additionally, 57.5% were concerned about and feared adverse events following immunization. Caregivers were found to have a low awareness about vaccination, and issues related to the organization of the health system were found to contribute towards MOV. Conclusions: One in three children experienced a MOV during a health service encounter. Potential interventions to reduce MOV include training of health workers about immunization policies and practices, improving caregivers' engagement and screening of vaccination documentation by health workers during every health service encounter. [ABSTRACT FROM AUTHOR]