Terri Hormon was served with the divorce papers and a restraining order late Monday night, reports CBS News Correspondent Bill Whitaker.
Kaine Hormon is said to have moved out of the house he shared with Terri. He reportedly took the couple's 18-month-old daughter with him.
Earlier Monday, Terri denied there were any problems with the marriage.
Police haven't officially named Terri a suspect or a person of interest, but say they gave her a second polygraph, reviewed her cell phone records, and examined her Ford truck.
Investigators say she's the last known person to have seen Kyron alive before he disappeared from his Portland school June 4. After a massive search, the Multnomah County sheriff's office recently described the case as a criminal investigation.
Police say Terri told them she took Kyron to school that morning. There was a science fair before classes began, and Terri took a photo of Kyron in front of his project.
She has told police she last saw him walking down a hallway toward his second-grade classroom, wearing a "CSI" T-shirt and dark cargo pants.
"The police are really; they think she is the heart of the investigation," observes former federal prosecutor Laurie Levenson.
All along, the family had appeared united, supporting TerriFriday. "I believe she's committed as the rest of our family is to finding Kyron," Kaine said at the time.
Kyron's biological parents also issued a new statement Monday - excluding Terri - saying they've been fully briefed on the criminal investigation and support the authorities, adding, "Any actions taken by the investigation, or by us, are based on the best interests of Kyron."
But, says Whitaker, their actions have many wondering whether an arrest might be imminent.