Some 1,200 trucks were stuck on Tuesday in a 15-km-long queue at the Batrovci-Bajakovo border crossing with Serbia, the European Union's new external frontier.
Drivers said they had to wait for 15 hours or more to cross.
Serbian customs and police officials claim the problem occurred following the harmonisation of Croatia's customs system with EU regulations.
There were no hold-ups at other border crossings with Serbia.
A statement from Croatia's customs service said problems were to be expected following the country's accession to the EU.
"It's all part of the adjustment," the statement said.
Passenger cars and buses were moving normally.
