On the E50/D5, which leads to the German city of Nuremberg, a queue of HGVs initially grew to over 20 kilometers long (12 miles), according to the motorway administration office and radio traffic news.
On the E55/D8 motorway connecting Prague with Dresden, trucks were queuing all the way back to the Czech town of Usti nad Labem on Monday morning.
Police were stopping drivers before letting them enter the tunnel there.
Czech authorities have boosted their testing capacity in border regions in response to the travel ban. This includes using the fire brigade to set up additional testing facilities.
Germany's border restrictions for Austria, Czechia:
Germany has stopped most land travel between regions in the east and south of the country and the neighboring Czech Republic and Austria's Tyrol, designating them "virus mutation areas."
In addition to HGV drivers, only returning Germans and essential workers such as doctors, can cross the borders.
Like the HGV drivers, they will also need a negative COVID-19 test, no older than 48 hours.
Around 1,000 police officers were mobilized on Sunday to ensure the new border controls.
State-owned rail company Deutsche Bahn suspended services to and from the affected areas.
The measures are set to remain in place for at least 10 days.
Border checks prompt backlash
Despite no reports of congestion at borders in Austria's Tyrol, the Austrian Foreign Ministry in Vienna summoned Berlin's ambassador to discuss the issue late Sunday.
"The measures have very severe consequences for the whole of Austria and therefore clearly contradict the lessons learned from last spring," Austrian Foreign Minister Alexander Schallenberg said.
Some carmakers have warned that the restrictions could once again bring factories to a standstill. The Czech Republic forms a key part of the supply chain for many German firms.
The DSLV, the federal association for logistics in Germany, said it was "extremely troubled" by the new border restrictions.
The German Association of the Automotive Industry (VDA) said supply chain disruption depended on the coronavirus test capacities at the borders. "We just have to wait and see" how the situation develops over the course of the day, a VDA spokesman added.
DW spoke with German Green Party MEP Jutta Paulus who also condemned the move, claiming that the situation could have been avoided if Germany had pushed for a unified EU strategy to combat the coronavirus pandemic in the latter half of 2020.
"I think [the] German presidency really did a bad job when in the last half year they did not even try to achieve a pan-European strategy with every member state more or less following the same rules, applying the same measures at the same case numbers," she told DW.
Germany defends measures
The German government rushed to defend the unpopular checks on Monday.
"We have a situation in which we had to take all the necessary steps to prevent the virus variants ... spreading as quickly in Germany as they are doing unfortunately in neighboring countries," Steffen Seibert, Chancellor Angela Merkel's chief spokesman, told a news conference.
Germany on Monday said it would not rule out expanding new travel controls to its French border with coronavirus variants surging in the Moselle region.
The suggestion has been met with disapproval in France.