Talking tough on crime with an election looming, Mr Trump vows to end the "bloodshed".
Chicago and two other Democratic-run cities are being targeted in the Republican president's move, amid a spike in violence.
But federal deployments in Portland, Oregon, have proved controversial. Local officials say they have raised tensions amid ongoing protests.
Law and order has become a key plank of Mr Trump's re-election bid in November.
Since the death on 25 May of an unarmed black man, George Floyd, in police custody in Minneapolis, Minnesota, there have been protests - sometimes descending into civil disorder - in scores of US cities.
Meanwhile, gun violence has spiked in metropolitan areas including New York City, Philadelphia, Los Angeles, Chicago and Milwaukee.
What is Operation Legend?
It is named after a four-year-old boy, LeGend Taliferro, who was shot dead while sleeping in his family home in Kansas City in June. The boy's mother joined the president at Wednesday's announcement.
The operation will see agents from the FBI, Marshals Service and other federal agencies work with local law enforcement, according to the US Department of Justice.
Mr Trump - whose opinion poll numbers have been slumping amid a coronavirus-crippled US economy - said: "This rampage of violence shocks the conscience of our nation."
The president, who accuses Democrats of being weak on crime, said: "In recent weeks there has been a radical movement to defend, dismantle and dissolve our police department."
He blamed this for "a shocking explosion of shootings, killings, murders and heinous crimes of violence". He added: "This bloodshed will end."
US Attorney General William Barr, who was with Mr Trump, said they had sent about 200 federal agents to Kansas City, Missouri. They would send a "comparable" number to Chicago and about 35 others to Albuquerque, New Mexico.
Mr Barr said the officers would be involved in "classic crime fighting", unlike the deployment of Department of Homeland Security agents which were sent to "defend against riots and mob violence" in Portland.
However policing in the US is the responsibility of states, and governors and locals officials have resisted the deployment of federal agents.
Oregon Governor Kate Brown has called it "a blatant abuse of power," and Portland's Mayor Ted Wheeler "an attack on our democracy."
Chicago Mayor Lori Lightfoot said on Tuesday: "We welcome actual partnership, but we do not welcome dictatorship."
What is going on in those cities?
On Tuesday night, federal agents fired tear gas, pepper balls and flashbangs at demonstrators in Portland, which has seen 54 consecutive nights of protests.
The officers used crowd-control munitions to disperse hundreds of people gathered outside a federal court.
The agents have been accused of driving in unmarked vehicles around Oregon's biggest city while wearing military fatigues and arbitrarily arresting a handful of demonstrators.
In the latest incident in Chicago, at least 14 people were shot outside a funeral home in a suspected gang-related attack.
The city has seen 34% more homicides than last year, according to the Chicago Sun-Times.
A 2018 FBI survey found the violent crime rate in Albuquerque was 3.7 times the national average. The rates of murder and rape were more than double the national average that year.
Kansas City, meanwhile, is on track to record its most homicides ever in 2020, according to the Kansas City Star. The city has now seen 110 homicides, with another 50 recorded across the greater metro area. BBC