Comparing Sunday's plebiscite to an earlier vote that preceded Moscow's annexation of Crimea, State Department spokeswoman Jen Psaki said: "This is the Crimea play-book all over again."
Kiev and its Western backers see Moscow's main aims as making sure the planned "referendum" goes ahead, and sabotaging all possibility of the nationwide presidential election two weeks later.
The West considers the May 25 presidential elections as crucial to hauling the former Soviet republic back from the brink.
Psaki also warned that "if Russia takes the next step" to move into eastern Ukraine and annex it, "harsh EU and US sanctions will follow."
More From This Section
Still, Putin has kept an estimated 40,000 troops on the border for the past two months.
Psaki's comments came as Russia rejected a new peace initiative amid mounting fears of open war.
Nearly 90 people have died in less than a week: half around the eastern town of Slavyansk, held by rebels since early April; and half in the southern port city of Odessa, where clashes culminated in a deadly inferno last Friday. Most of those killed have been pro-Russian fighters and activists.