Distinct 18O depletion is characteristic of a large majority of the 620–550 Ma felsic igneous rocks of Avalonia in the northern Appalachian orogen. Neoproterozoic rocks in the Boston Avalon terrane have the lowest δ18OWR values (≥–3.1‰), followed by the Mira terrane in Cape Breton Island and the Caledonia terrane in New Brunswick (≥–1.2‰), the Avalon terrane in Newfoundland (≥+2.8‰), and the Antigonish Highlands in Nova Scotia (≥+5.3‰). In contrast, this depletion of 18O is observed in very few of the Paleozoic felsic igneous rocks from these Avalonian terranes, and also in very few of the Neoproterozoic and Paleozoic felsic igneous rocks from the inboard Ganderian terranes. The low-18O character of the Neoproterozoic igneous rocks is related to regional pervasive, post-magmatic alteration by predominantly meteoric-hydrothermal fluids (δ18OH2O ∼–6‰ to –4‰) at 200–450 °C. The alteration likely occurred during late Neoproterozoic transtensional extension of Avalonia. Large-scale fluid infiltration and circulation within the Avalonian crust accompanied this extension with development of pull-apart sedimentary basins and extension-related magmatism that were the prelude to Cambrian submergence of Avalonia. This regional 18O depletion provides a geochemical fingerprint by which Avalonia can be distinguished from other peri-Gondwanan terranes. These data suggest that Avalonia existed as a composite terrane on the Gondwanan margin in the Neoproterozoic, separate from Ganderia.