Ann Serrano Lopez filed her petition, citing irreconcilable difference, on Tuesday in Los Angeles.
The pair announced their breakup in September and said they would remain partners in a charitable foundation. They have a 14-year-old daughter, and Ann Lopez is seeking physical custody.
The filings do not offer any additional details about the split.
The pair were married in September 1993 and did not list a separation date.
The filing was first reported by celebrity website TMZ.
The 49-year-old comedian hosts the talkshow "Lopez Tonight" on TBS.