A total of 14 crash deaths have been reported in those Tesla investigations, including the most recent three.
Of the 35 special crash investigations by the NHTSA into Tesla since 2016 involving advanced driver assistance systems, Autopilot use has been ruled out in three.
NHTSA separately said on Wednesday that in April it had opened another special crash investigation involving a 2016 Tesla Model X in Florida that resulted in a minor injury.
NHTSA did not immediately identify the crash that killed three in the new probe but the Orange County Register reported a May 12 Tesla Model S crash killed three occupants and injured three workers were when it struck construction equipment along Pacific Coast Highway in Newport Beach.
Tesla's Autopilot and other driver assistance systems are coming under increased scrutiny. Tesla says on its website that Autopilot enables vehicles to steer, accelerate and brake automatically but "require active driver supervision and do not make the vehicle autonomous."
In August, NHTSA said it opened a formal preliminary evaluation into Autopilot and has identified at least a dozen crashes involving Tesla models and emergency vehicles. That investigation is still pending.
Tesla did not immediately respond to a request for comment.