The operation was the largest worksite enforcement by U.S. Immigration and Customs Enforcement, or ICE, since Republican President Donald Trump took office last January, agency spokeswoman Danielle Bennett said in an email.
"Today's actions send a strong message to U.S. businesses that hire and employ an illegal workforce: ICE will enforce the law, and if you are found to be breaking the law, you will be held accountable," ICE Deputy Director Thomas Homan said in a statement.
Taking a harder line on illegal immigration, including building a wall at the border with Mexico, was a touchstone for Mr Trump during the 2016 election campaign.
At a White House meeting on Tuesday, Mr Trump urged lawmakers to quickly reach a bipartisan deal on a program for "Dreamers," people who came to the country illegally as children, before moving on to a comprehensive immigration bill.
"Notices of inspection" were delivered on Wednesday to 98 7-Eleven stores in 17 states and the District of Columbia beginning at 6 a.m. in each local time zone. Owners and managers have three business days to produce documents showing their employees are in the country legally or they could face civil and criminal penalties, ICE said.
The states where the employment audit notices were served were California, Colorado, Delaware, Florida, Illinois, Indiana, Maryland, Michigan, Missouri, Nevada, New Jersey, New York, North Carolina, Oregon, Pennsylvania, Texas and Washington, ICE said.
Based in Irving, Texas, 7-Eleven has 60,000 convenience stores in 18 countries, including 8,500 in the United States, according to its website.
The federal operation was a follow-up to the 2013 arrests of nine 7-Eleven franchise owners and managers, ICE said in a statement. Those owners were accused of hiring employees living illegally in the United States and giving them identities stolen from U.S. citizens.
The 21 people who were "administratively arrested" on Wednesday on suspicion of being in the country illegally were given notices to appear in immigration court and could be deported.
(Reporting by Bernie Woodall in Fort Lauderdale, Fla.; Editing by Colleen Jenkins and Peter Cooney)