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The Port of London Authority is working with police to identify the source of a laser that was shone at a launch vessel.
The public trust has said the devices can permanently damage vision, while endangering those travelling or working on the Thames.
Tougher laser misuse laws came into force in July last year - with offenders now facing up to five years in prison and unlimited fines.
The legislation removes the need for police to establish proof of intention to endanger, making it harder for culprits to claim their actions were accidental.
Martin Garside, a spokesman for the Port of London Authority, confirmed the incident near Gravesend on the evening of Tuesday, January 8.
"No one was injured on this occasion, but lasers can cause very serious damage to the eye – including permanent damage to vision - and also endanger safety on the river.
"We are investigating the incident and liaising closely with the police – both on land and water – to identify the exact location of the perpetrator."
Alan Carr, a volunteer crew member for the Royal National Lifeboat Institution (RNLI), has said shining lasers could also slow down a rescue mission.
The 46-year-old from Greenhithe, who is based at Gravesend Lifeboat Station, said: "If a laser was shone in someone's eyes, they would have no ability to drive any more.
"It could impact a search for someone, as we'd be helping the driver of the boat, and couldn't go ahead until the crew member had been looked at."
He added that, while it is possible to turn away from a laser beam, boats can only drive through certain areas of the river.
The Laser Misuse (Vehicles) Act also applies to planes, trains and cars.
Kent Police spokesman Vicki Foster said: "Police received a call at 5.04pm on Tuesday, January 8 to a report of a laser being shone once at a boat in the water near Gravesend.
"The caller was unsure of the location as to where the laser had come from and appropriate advice was provided to the caller following the incident."