Carla del Ponte said on Wednesday that the Serbian city had become a "safe haven" for fugitives from her tribunal.
But the Serbian government immediately denied all knowledge of the whereabouts of Karadzic, the Bosnian Serb leader during the bloody 1992-1995 war in Bosnia-Hercegovina.
Del Ponte said Karadzic had apparently joined his then army chief Ratko Mladic in the Serbian capital, where authorities have frozen contacts with her.
"Karadzic is now residing in Belgrade," said the chief prosecutor from the Hague-based International Criminal Tribunal for the former Yugoslavia (ICTY), citing a "credible source" who came forward with information last week.
Indicted by the UN war crimes court for genocide for his role in Bosnia's war, Karadzic remains for many the symbol of the worst atrocities perpetrated in Europe since World War II, but Serbs in Bosnia consider him a war hero.
"I don't know how we can get those (fugitves in Serbia) arrested and transferred to The Hague. But we are expecting... that the tribunal will not close the door without having Mladic and Karadzic in The Hague"
Carla del Ponte,
International Criminal Tribunal for the former Yugoslavia
The NATO-led Stabilization Force in Bosnia has recently stepped up efforts to find Karadzic, who tops the list of war crimes suspects along with Mladic.
Del Ponte, speaking after talks with EU foreign policy chief Javier Solana in Brussels, said: "I received just last week information from a credible source that... Karadzic is now in Belgrade."
But Serbian Prime Minister Zoran Zivkovic flatly denied any knowledge of Karadzic's whereabouts.
"Her statements are nothing new or unexpected," Zivkovic was quoted as saying by the Beta news agency.
"She has been saying the same or similar things for years but so far she has not provided any information which could help arrest the indictees, and even less to support her claims.
"State autorities have no information which could support her claims."
NATO this month vowed no let-up in the manhunt for Karadzic, after a near-miss by alliance forces was reported.
And NATO spokesman James Appathurai called for Belgrade to cooperate with the ICTY.
"Each government has a responsibility to cooperate with the tribunal," he said, noting that such cooperation was a condition for Serbia's hopes to join NATO's Partnership for Peace (PFP) programme.
Del Ponte also lamented that cooperation between the ICTY and Serbian authorities had ground to a halt.
"Her statements are nothing new or unexpected. She has been saying the same or similar things for years but so far she has not provided any information which could help arrest the indictees, and even less to support her claims"
Serbian Prime Miniser
"We must say at this time cooperation is frozen," she said, adding "Belgrade is now a safe haven for fugitives".
And she said around 15 war crimes suspects remained on the run in Serbia.
"I don't know how we can get those arrested and transferred to The Hague. But we are expecting... that the tribunal will not close the door without having Mladic and Karadzic in The Hague."
The charges against Karadzic and Mladic relate in particular to the three-and-a-half-year-long siege of Sarajevo, which killed around 10,000 civilians.
Karadzic is also accused of being one of the prime organizers of the 1995 massacre of more than 7000 Muslim men and boys in the eastern Bosnian town of Srebrenica.