The film was found by Ray Begovich, a journalism professor at Franklin College in Indiana, who was watching hours of unedited World War II film footage.
Begovich found the film while visiting the National Archives in College Park, Md., doing research for a biography on Roosevelt's director of war information.
The 16-millimetre eight-second film showed Roosevelt, the 32nd President of the US (1933-1945), visiting the Navy's US heavy cruiser Baltimore on July 26, 1944.
Toward the end of the film, a photographer captured Roosevelt exiting a doorway down a ramp. The president moved in a gliding motion, the top of his head barely visible, as he was in a wheelchair.
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Begovich watched the clip dozens of times before requesting a DVD copy. Over the next several years, he showed the footage to colleagues and experts, and conducted extensive online searches.
His hunch was right: The clip appears to be the only known footage of the president in his wheelchair to surface in the decades since Roosevelt's death in 1945. Begovich kept largely quiet about his discovery until this week.
"But it's definitely very rare," she said.
In 1921, at age 39, Roosevelt contracted polio. The illness left him paralysed below the waist.
At the time, people affected by polio were often kept out of sight. As Roosevelt advanced in his political career, he carefully concealed his condition from the public. He used leg braces, a cane or the help of a companion's arm to walk short distances during public appearances, the report said.
Begovich has a theory about who took the photograph -- he suspects a US Navy photographer, impressed by a presidential visit to the base during wartime. The way the footage breaks away suggests the person filming knew it was taboo to do so, he said.
Begovich said he hoped his accidental discovery would inspire scholars to look more intently for surviving film footage of Roosevelt -- and help bolster people with disabilities who want to seek political careers.