Seddique Mateen's past as a source for federal law enforcement emerged at the trial in Orlando of Noor Salman, 31, Omar Mateen's widow, who prosecutors allege had prior knowledge of her husband's plans.
Omar Mateen died in the June 12, 2016 mass shooting at the Pulse nightclub, which he claimed to have carried out on behalf of the Islamic State group.
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Defense attorneys, in documents filed with the court over the weekend, called for a mistrial in Salman's case following the revelation that her father-in-law had served as an FBI informant.
But US District Judge Paul Byron rejected the request on Monday, the Orlando Sentinel newspaper reported.
According to Salman's lawyers, the US Attorney's office acknowledged in an email over the weekend that Seddique Mateen was an "FBI confidential human source at various points in time between January 2005 and June 2016."
In addition, it said, Seddique Mateen was the subject of an investigation for money transfers he had made to Turkey and Afghanistan.
The FBI also said it had received a tip that Seddique Mateen was engaged in fund-raising that may have been intended to contribute to "an attack against the government of Pakistan."
The defense attorneys said the government's failure to disclose the information violated Salman's "right to a fair trial" and called for the charges against her to be thrown out.
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"If the government had provided this information, the defense would have investigated whether a tie existed between Seddique Mateen and his son, specifically whether Mateen's father was involved in or had foreknowledge of the Pulse attack," the defense said.
Seddique Mateen, who is originally from Afghanistan, was on the government's initial witness list but was finally not called to testify.
Judge Byron dismissed the request for a mistrial.
"This trial is not about Seddique Mateen," the Orlando Sentinel quoted the judge as saying. "It's about Noor Salman."
Salman is facing charges of providing support to a foreign terrorist organisation and obstructing justice.
Defense lawyers have told the court she was in an abusive relationship with Omar Mateen and had no knowledge of his plans.
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