Despite favourable conditions times were slightly slower than the weekend, with riders and teams instead focusing on various development and set-up work.
With around three hours to run Crutchlow, who claimed his first MotoGP podium on Sunday, moved up into top spot with a 1m56.461s that was never headed.
"It was a good test and I was really pleased with my times," said Crutchlow.
"We tried some different settings but the only problem I have with this bike now is we make some big changes but the lap-time is the same, so you never know which direction to go in.
"In practice for the race on Friday I struggled with the hard tyre but today I tested it again and I was lot faster and consistent as well, so that was a big positive.
"We can leave Brno really happy because it has been a great weekend. I signed a new contract with the Monster Yamaha Tech 3 Team, got my best grid position, scored my first ever MotoGP podium and then set the fastest time at the test today.
"We can go to Misano in a positive frame of mind and I'm confident we will be challenging close to the front again."
His Tech 3 Yamaha team-mate Andrea Dovizioso backed him up in second, just over 0.1s down the road, while behind them Yamaha's Jorge Lorenzo and Honda's Dani Pedrosa once again proved almost inseparable in third and fourth.
Lorenzo, who tested both this year's bike and a new M1 prototype frame fitted with his current engine, wound up third fastest.
He ended just four hundredths of a second clear of Pedrosa, whose work was based around testing new parts. Honda plans to test further parts at Aragon next week.
On the other side of the Honda garage, Jonathan Rea got his first taste of 1000cc MotoGP machinery as he prepares to stand in for the injured Casey Stoner at Misano and Aragon.
The World Superbike racer reported that adjusting to Bridgestone rubber was the hardest part of the day, but nevertheless improved consistently and eventually ended the day eighth fastest.
The two works Hondas were separated by the satellite counterparts of Stefan Bradl and Alvaro Bautista, and the second Yamaha of Ben Spies.
MotoGP safety officer Loris Capirossi meanwhile tested numerous bikes, including Bautista's and Colin Edwards' Suter BMW in order to get a feel of how different bikes reacted to the tyres.