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Graphical abstract Highlights • Fractionation of sorghum stems yields fractions exhibiting diverse properties. • Physical fractionation by wet milling and sieving can yield pith-enriched fractions. • Sorghum pith cell walls are low in lignin and more readily digestible by enzymes. Abstract Sorghum offers enormous potential as a feedstock for the production of fuels and chemicals from both water-extractable sugars and the cell wall biopolymers, while its within-plant structural and compositional heterogeneity may allow for physical fractionations to tailor feedstock properties to a biorefining process. In this study, the stem internodes of two sorghum (Sorghum bicolor L. Moench) genotypes, a sweet sorghum ('Della') and a forage/energy sorghum ('TX08001'), were first subjected to fractionation by manual classification by stem anatomy and internode proximity to the ground to yield 18 fractions. These fractions exhibited substantial differences in cell wall morphology, composition, and recalcitrance to mild alkaline pretreatment and enzymatic hydrolysis. While the sweet sorghum cultivar held nearly 70% more water-extractable sugar (sucrose, glucose, fructose, starch) in the stems than the forage/energy sorghum hybrid, both cultivars exhibited comparable diversity of composition and these compositions were remarkably similar in similar tissues and stem regions between the two cultivars. The fractions isolated from the pith parenchyma were the least recalcitrant to mild alkaline pretreatment and enzymatic hydrolysis and contained less lignin than fractions isolated from the epidermis, outer and inner rind, and internal vascular bundles. The pith samples isolated from the lowest region of the stem from both cultivars exhibited near-theoretical sugar hydrolysis yields when no pretreatment was employed and exhibited the lowest lignin contents of any of the fractions. Next, a physical fractionation approach approximating a commercial "de-pithing" process utilizing wet disintegration and sieving was applied to the forage/energy sorghum. A pith-rich fraction representing approximately 20% of the extractives-free mass of the stem could be isolated with this approach and, relative to the other fractions, was low in lignin, high in ash, highly hygroscopic, and showed an improved response to mild alkaline pretreatment and enzymatic hydrolysis at low enzyme loadings. Overall, these results demonstrate how heterogeneity within sorghum stems can be exploited using physical fractionation approaches to yield fractions enriched in desired properties that may allow for more streamlined processing. [ABSTRACT FROM AUTHOR]