Owen Chamberlain was born in San Francisco on July 10, 1920. His father was W. Edward Chamberlain, a prominent radiologist with an interest in physics. His mother's maiden name was Genevieve Lucinda Owen.
He obtained his bachelor's degree at Dartmouth College in 1941. He entered graduate school in physics at the University of California, but his studies were interrupted by the involvement of the United States in World War II. In early 1942 he joined the Manhattan Project, the U.S. Government organization for the construction of the atomic bomb. Within the Manhattan Project he worked under Professor Emilio Segrè, both in Berkeley, California, and in Los Alamos, New Mexico, investigating nuclear cross sections for intermediate-energy neutrons and the spontaneous fission of heavy elements. In 1946 he resumed graduate work at the University of Chicago where, under the inspired guidance of the late Professor Enrico Fermi, he worked toward his doctorate. He completed experimental work on the diffraction of slow neutrons in liquids in 1948 and his doctor's degree was awarded in 1949 by the University of Chicago.