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Silicon is a beneficial element for many plants and is deposited in plant tissue as amorphous bio-opal called phytoliths. The biochemical processes of silicon uptake and precipitation induce isotope fractionation: the mass-dependent shift in the relative abundances of the stable isotopes of silicon. At the bulk scale, delta Si-30 ratios span from -2 to +6 parts per thousand. To further constrain these variations in situ, at the scale of individual phytolith fragments, we used femtosecond laser ablation multi-collector inductively coupled plasma-mass spectrometry (fsLA-MC-ICP-MS). A variety of phytoliths from grasses, trees and ferns were prepared from plant tissue or extracted from soil. Good agreement between phytolith delta Si-30 ratios obtained by bulk solution MC-ICP-MS analysis and in situ isotope ratios from fsLA-MC-ICP-MS validates the method. Bulk solution analyses result in at least twofold better precision for delta Si-30 (2s on reference materials <= 0.11 parts per thousand) over that found for the means of in situ analyses (2s typically <= 0.24 parts per thousand). We find that bushgrass, common reed and horsetail show large internal variations up to 2 parts per thousand in delta Si-30, reflecting the various pathways of silicon from soil to deposition. Femtosecond laser ablation provides a means to identify the underlying processes involved in the formation of phytoliths using silicon isotope ratios.