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Studies of plant–microbe interactions using synthetic microbial communities (SynComs) often require the removal of seed-associated microbes by seed sterilization prior to inoculation to provide gnotobiotic growth conditions. Diverse seed sterilization protocols have been developed and have been used on different plant species with various amounts of validation. From these studies it has become clear that each plant species requires its own optimized sterilization protocol. It has, however, so far not been tested whether the same protocol works equally well for different varieties and seed sources of one plant species. We evaluated six seed sterilization protocols on two different varieties (Sugar Bun and B73) of maize. All unsterilized maize seeds showed fungal growth upon germination on filter paper, highlighting the need for a sterilization protocol. A short sterilization protocol with hypochlorite and ethanol was sufficient to prevent fungal growth on Sugar Bun germinants; however a longer protocol with heat treatment and germination in fungicide was needed to obtain clean B73 germinants. This difference may have arisen from the effect of either genotype or seed source. We then tested the protocol that performed best for B73 on three additional maize genotypes from four sources. Seed germination rates and fungal contamination levels varied widely by genotype and geographic source of seeds. Our study shows that consideration of both variety and seed source is important when optimizing sterilization protocols and highlights the importance of including seed source information in plant–microbe interaction studies that use sterilized seeds. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .