Wheelbarger's announcement marks the first time the United States has included any such condition to its sale of Patriot missile defence systems to foreign states.
Turkey's President Recep Tayyip Erdoğan confirmed once again during a live interview on Turkish television last week that the purchase of S-400 batteries from Russia is a done deal and there is no turning back.
Erdoğan also said Turkey wıll seriously consider buying S-500s, the new generation of Russian defense systems. Turkish officials say that the S-400s will be co-producted by two nations.
Wheelbarger called Turkey a "critical ally in NATO's southern flank" at the National Security Challenges and U.S. Military Activities in Europe hearing, where she appeared alongside General Curtis M. Scaparrotti, the top U.S. general in Europe.
Wheelbarger said during her testimony that Ankara's contributions to the coalition missions in Afghanistan, Iraq and Kosovo would continue. The top Pentagon official added that the United States continues "to reiterate the importance of Turkey remain grounded in the NATO security structures."
Turkey's close relationship with Russia has prompted many Western officials and analysts to comment on the possibility of a Turkish split from NATO. However NATO does not have any withdrawal clause and there is no mechanism to kick out members as no member so far has left the Western military alliance.
Wheelbarger said Washington had been "clear expressing its concern about Turkey's stated intent of procuring the S-400 from Russia, which would introduce risks to U.S. and NATO defence technologies."
U.S. officials have argued the S-400 batteries, which would be reportedly deployed around Ankara, could potentially be used by Russia to gather information on the new generation U.S. fighter F-35 fighter jets.
Wheelbarger closed the Turkey reference in her testimony by introducing a new condition for the U.S. sale of Patriots to Turkey never voiced by Washington until Wednesday's hearing.
"We thank Congress for its support for offering the Patriots to Turkey as an alternative to the S-400. We will only continue to discuss the potential sale with Turkey if Turkey commits to not accepting the S-400," she said.