Gayle Nix Jackson is looking to either get the film back or get $10 million in compensation over the film shot by Orville Nix in November 1963.
Orville Nix sold the film to a news agency in 1963 but it was later handed to the government for its inquiries.
Gayle Nix Jackson says she was told this year the government agency believed to be in possession did not have the film.
The 8mm film was shot from the opposite side of the presidential limousine from where the famous Zapruder film was taken on November 22, 1963.
The Nix film shows the bullet hitting JFK, First Lady Jackie Kennedy climbing on to the boot of the limousine, and secret service agent Clint Hill jumping into the vehicle.
The film is shot from Dealey Plaza, showing Abraham Zapruder across the street and the famous grassy knoll, from where some witnesses thought they heard a shot fired.
It is not as complete as the Zapruder movie, as it shows only part of the assassination.
However, the lawsuit cites the Warren Commission – which investigated the assassination – as saying the Nix film was “nearly as important as the Zapruder film”.
The government purchased the Zapruder film for $16 million in 1999 in a settlement with his heirs.
Orville Nix sold his film to UPI for $5,000 with an agreement for its return after 25 years.
But the film was handed over to the government for the inquiry.
Its last known sighting was with the House Select Committee on Assassinations in 1978.
The film was believed to have been handed to the National Archives and Records Administration, but the lawsuit says the agency has told Gayle Nix Jackson, who lives in Fort Worth in Texas, it no longer has the movie.
Gayle Nix Jackson told the Associated Press it was incomprehensible authorities would lose “an important piece of historical evidence”.
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