Kiev's military said insurgents using Russia-supplied weapons shelled adults and children in a convoy with white flags on a road from the restive city of Lugansk, leading to "dozens of dead".
The allegations came after five hours of crisis talks in Berlin brought no consensus on how to end the conflict, and President Petro Poroshenko said Kiev was pressing on with its drive to oust rebels, having "laid siege to cities most controlled" by them.
"The convoy had white flags and were marked as civilian," he said at a briefing. Kiev believes it was shelled from mortar guns and Grad rocket systems supplied from Russia and was "completely destroyed."
"We ask that any videos from the scene are not released to the public, because they are atrocious," he added.
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Lugansk has been the hardest-hit city still controlled by rebels, where water and power have been cut off for more than two weeks.
"Not a single convoy of refugees was shot at in the Lugansk region," he told journalists at a briefing.
A marathon meeting in Berlin between the foreign ministers of Ukraine, Russia, France and Germany broke up without agreement on how to end more than four months of conflict that has killed over 2,100 people and left the region facing a humanitarian catastrophe.
Moscow has demanded that Ukraine government forces halt an offensive against main separatist strongholds, while Kiev accuses Russia of pouring in more arms to save the unravelling insurgency.
The Ukrainian presidency said this evening that rebel boasts of receiving Russia-trained fighters and various weaponry including tanks forced Kiev to "look at the military operation in new light" and "continue the offensive".