Sweet sorghum is one of the important crops that has been widely reported to be recalcitrant to genetic manipulation endeavors. It can serve as biofuel, food, and food per production systems toward energy sources as well as human and animal sustenance. To deploy biotechnology tools in advancement of this crop of such agronomic importance, genetic and molecular investigation for understanding is necessary. Underdeveloped cell walls are a significant physiochemical state for microspores per their developmental stages, which makes it ideal for DNA isolation, being readily lysed as natural protoplasts. After harvesting panicles from the plants grown in controlled greenhouse conditions those are separated into sections per developmental stages is the first step. During micro-gametogenesis the unicellular microspore divides asymmetrically to subsequently give rise to a mature pollen grain with a vegetative and generative cell, while the absence of the participation of the anther wall in pollen formation makes it possible to investigate these developmental events directly. Thereby, from distal to basal end along panicle length, the five stages—mid-binucleate, early-binucleate, late-uninucleate, mid-uninucleate, and early-uninucleate microspores—can be yielded toward fractionalization. Whole-genome amplification is ideally achieved through individually isolated microspores with underdeveloped exine from anthers obtained after aseptic handling of spikelets using 75% ethanol and 1% sodium hypochlorite as sterilizing agents. The free-floating microspores, thus separated from the surrounding anther wall tissues, yield single gametophyte-based high-quality genomic DNAs. Efficient breeding of sweet sorghum through genetic tools can be achieved through free microspore release after aseptic isolation and whole-genome amplification.