Background: Tobacco retailers have an important role to play in reducing access to tobacco among youth, but in the USA, their role has become more complicated because of recent developments in policies and industry, such as the Indiana Tobacco-21 law and the rising popularity of electronic nicotine delivery systems (ENDS) products. This study aimed to explore Indiana retailers' knowledge, attitudes, and skills related to the prevention of underage sales of tobacco and ENDS products and identify retailers' needs for further education and training. Methods: The study involved a secondary analysis of anonymous cross-sectional data from the 2020 Indiana Tobacco Compliance Survey conducted by the Indiana Division of Mental Health and Addiction (DMHA). Managers and owners of retail stores who sold tobacco or ENDS products (i.e., large corporate-owned stores and smaller venues such as tobacco shops, vapour shops, and convenience stores) were eligible for inclusion. Records of retailers' tobacco certificates were utilised for random sampling. Subgroup comparisons were conducted using chi-square tests. Results: The survey sample (n = 102) included participants from all 10 DMHA planning regions in Indiana and all types of tobacco retail stores. Many retailers did not know about or understand the negative impacts of ENDS products on young people or existing regulations to minimise these impacts and the Indiana State Tobacco-21 law. Retailers in larger stores had a greater objective knowledge and reported more frequent prevention programme utilisation than smaller stores (e.g., by means of 'We Card'). Conclusions: Underage tobacco sale prevention procedures varied significantly, but resources such as 'We Card' and 'This is Our Watch' were more popular than alternatives. Retailers reported a need for more convenient education and training options on how to check photo identification and refuse sales to minors.