We describe a simple single‐pot method for collection and preparation of natural water for microplastic analyses. The method prepares samples in the same vessel (mason jars) that they are collected in right up until the microplastics are transferred onto filters or spectroscopic windows for analyses. The method minimized contamination, degradation, and losses, while increasing recoveries and throughput when compared with conventional sieving. We applied it to surface grab samples collected from the Mississippi River and its major tributaries during and after historic flooding in 2019. Microplastics (>~30 µm) were quantified using Nile red fluorescence detection, and a small subset of samples were identified by micro‐Fourier transform infrared imaging spectroscopy. Concentrations were lower during the flooding, likely due to dilution. Concentrations ranged from approximately 14 microplastics/L in the Tennessee River during flooding to approximately 83 microplastics/L in the Ohio River during low‐flow (summer) conditions. Loads of microplastics tended to increase downriver and ranged from approximately 87 to approximately 129 trillion microplastics/d near New Orleans. Most of the microplastics (>60%) were in the lower size fraction (~30–90 µm) and consisted primarily of fragments (~85%), followed by fibers (~8%) and beads (~7%), with polyester, polyethylene, polypropylene, and polyacrylate as the primary microplastic types. Overall, we demonstrate that the single‐pot method is effective and versatile and, because it uses relatively inexpensive and easily assembled materials, can be adapted for microplastic surveys worldwide, especially those involving sample collection by volunteers from the community and schools. Environ Toxicol Chem 2020;39:986–995. © 2020 SETAC