The vandalism happened outside the Alibi Lounge on Adam Clayton Powell Boulevard just before 1 a.m. Monday.
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Surveillance video showed a man sitting on an adjacent stoop, lighting the flag in several places before walking off.
Other video showed charred pieces of the flag on the ground outside the bar. No one was injured.
The incident came a little more than a month after a man was caught on camera setting two flags on fire.
Someone in the community donated two massive rainbow flags to the bar to replace them, and now, one of those has gone up in flames too.
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The bar's owner told Eyewitness News no one saw the incident happen.
Police were reviewing surveillance video and said a tip led them to the suspect arrested in the case.
Authorities say 20-year-old Tyresse Singleton is charged with two counts of fourth-degree criminal mischief as a hate crime and two counts of fifth-degree arson.
"I'm relieved to know that the person who burned the flag twice and appeared to be the same individual has been arrested and that hopefully it means we can move forward," said the owner of the bar, Alex Minko.
Governor Andrew Cuomo has directed the State Police Hate Crimes Task Force to assist in the investigation.
"I am appalled by the burning of yet another pride flag outside an LGBTQ bar in Harlem," he said. "This act of hate is repugnant to our values of equality and inclusion, and we will not stand by and allow these cowardly acts to continue in New York.'
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