"We once again urge Iran's leadership to quickly resolve all outstanding American citizen cases," Clinton said.
The increased emphasis on Americans in Iran came after news that a graduate student from the United States who had been held in Iran returned this week to Los Angeles.
The student, Esha Momeni, was imprisoned for a month last year because of her research on the Iranian women's rights movement. She was freed in November but was only now able to return to the U.S.
Retired FBI agent Robert Levinson has been missing since March 2007. Three hikers -- Joshua Fattal, Shane Bauer and Sarah Shourd -- were detained by Iranian authorities on July 31. And an Iranian-American scholar, Kian Tajbakhsh, was arrested last month on charges related to provoking unrest.
"Our goal is to ensure the safe return of all our missing or unjustly detained American citizens to the United States as quickly as possible so that they can be reunited with their families," Clinton said in her statement.
The United States and Iran have not had formal diplomatic relations since the 1979 Islamic Revolution, but in March, Clinton took the unusual step of sending a letter asking the Iranian government's permission for Momeni, Levinson and Iranian-American journalist Roxana Saberi.
Saberi was convicted of espionage before being released on an appeal in May. Levinson was last seen on Iran's Kish Island on March 8, 2007. He disappeared in Iran while investigating cigarette smuggling for a client of his private security firm.
The three hikers are accused of crossing over the border from Iraq, though they have said it was by accident and they had no intention of straying into Iran.
The Associated Press contributed to this report.