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Well-aligned, densely packed RuO2 nanocrystals (NCs) have been grown on sapphire (SA), LiNbO3 (LNO), and LiTaO3 (LTO) substrates with different orientations via reactive magnetron sputtering using a Ru metal target. The surface morphology and structural and spectroscopic properties of the as-deposited NCs are characterized using field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and micro-Raman spectroscopy (RS). FESEM micrographs reveal that NCs grown on SA(100)/LNO(100) are vertically aligned, whereas the NCs on SA(012)/LTO(012) and SA(110) contain singly and doubly tilted alignments, respectively, with a tilted angle of ∼35° from the normal to the substrates. NCs grown on SA(001) show in-plane alignment with the mosaic structure. The XRD results indicate that the NCs are (001), (101), and (100) oriented on SA(100)/LNO(100), SA(012)/LTO(012)/SA(110), and SA(001) substrates, respectively. A strong substrate effect on the alignment of the RuO2 NCs deposition has been demonstrated. XPS analyses reveal the coexistence of higher oxidation states of Ru in the as-deposited RuO2 NCs. The Raman spectra show the red-shift and asymmetric peak broadening of the RuO2 signatures with respect to that of the bulk counterpart, which are attributed to both the size and residual stress effects, whereas the intensity of certain modes follows the selection rules for the different oriented NCs.