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Abstract Premise Sorghum is a multi‐use crop, the efficient breeding of which requires the development of new genetic tools. One such tool could be the genetic assessment of free microspores, which are released just after the tetrad stage of pollen development. Microspores are ideal for DNA isolation as they have underdeveloped cell walls and can be readily lysed as natural protoplasts. Methods Four cultivars of Sorghum bicolor (ʻAchi Turi’, ʻDale’, ʻLocal’, and ʻTopper 76‐6’) were grown in a greenhouse until flowering (7.7–11.5 cm flag leaf internode length), after which 30 immature microspores were isolated from each line. Plant height, time to flowering, boot radius, and spikelet maturation were recorded for each cultivar. The exine development of the microspores was observed under an inverted Nikon microscope, and those with underdeveloped exine were subjected to whole‐genome amplification and sequencing. Results Microspores in the early uninucleate to early binucleate stages had underdeveloped exine, and were therefore ideal for DNA extraction. High‐quality DNA was obtained from these single‐cell gametophytes. The average DNA concentration was 2902 ng/µL, with fragment sizes comparable to those obtained from leaf tissue extractions. Discussion Harvesting panicles with immature microspores means the entire gametic population is accessible for DNA analyses. This is the first amplification of whole‐genome DNA fragments from sorghum single‐cell microspores isolated during gametogenesis.