The roads were blocked with heavy vehicles and trucks on Sunday, a day after Serbia's President Aleksandar Vucic said he would ask the NATO-led peacekeeping force in Kosovo to permit the deployment of 1,000 Serbian troops in the Serb-populated north of Kosovo.
The roadblocks, which Serbs say were erected to protest the recent arrest of a former Kosovo Serb police officer, came despite the postponement of the December 18 municipal election opposed by Kosovo Serbs.
Mr Vucic said that his message to the Serbs in Kosovo is that "there is no surrender and there will be no surrender."
He said the Serbs had been forced to erect the barricades to protect themselves from Kosovo security forces.
Kosovo Prime Minister Albin Kurti accused Belgrade of trying to destabilise Kosovo.
He said Serbia also is trying to bring an end to the EU-mediated dialogue on normalising bilateral ties and take it to the United Nations Security Council, where Belgrade hopes to get support from Russia and China.
Mr Kurti called on Kosovo's Serbs "to distance themselves from the criminal groups and Mr Vucic's regime that is funding them and looking for a war".
The European Union rule of law mission, known as EULEX, reported that a flash grenade was thrown at an EULEX reconnaissance patrol overnight. There were no injuries or damage.
EULEX, which has some 134 Polish, Italian and Lithuanian police officers deployed in the north, called on "those responsible to refrain from more provocative actions" and said it urged the Kosovo institutions "to bring the perpetrators to justice."
European Union's high representative, Josep Borrell, said the EU "will not tolerate attacks on EULEX or use of violent, criminal acts in the north."
"Barricades must be removed immediately by groups of Kosovo Serbs," he said on Twitter.
"Calm must be restored … all actors must avoid escalation."
Unidentified masked men were seen on the Serb barricades that blocked main roads leading to the border with Serbia, as Kosovo authorities closed two border crossings to all traffic and pedestrians.
An increased presence of Kosovar Albanian police in areas with a mixed population, as well as more international police and soldiers, were seen in the north on Sunday.
Serbian officials said heavily armed Kosovo policemen "burst into" the premises of a strategic Serb-controlled dam on the artificial Gazivode Lake on the border with Serbia. The report could not be independently verified.
The Serb officials asked for urgent EU action to "restrain the thugs" who, they alleged, want to trigger another war with Serbia.
Interior Minister Xhelal Zvecla, using the Albanian name for the lake, said that "situation at the Ujmani Lake is under control," adding that there was an exchange of fire overnight.