The seven-time MotoGP champion found himself struggling on the Losail International Circuit, finishing 0.881s behind Marc Marquez's Honda.
Rossi attributed his problems to his bike's back tyre which he struggled to get any grip on.
"Today was difficult," Rossi said in the Losail paddock. "I wasn't fast enough and my pace was not strong. We tried to improve the rear because also in the test here we suffered with rear grip, but we 'damaged' the tyre from the beginning.
"It looks like we are not okay with the setting because Jorge [Lorenzo, fourth fastest] is able to use the tyre in a better way and did a good pace. This is good because it means the bike is not so bad. But for sure the Hondas are very fast.
"I don't know exactly [what is wrong] but it seems we don't make the rear tyre comfortable. When you don't use it in a good way the tyre starts to damage and then the bike moves and it is very difficult."
Rossi, who is starting his 13th MotoGP season, was also disappointed that there is only one more practice session remaining before qualifying kicks off.
"The shame now is that we have just one practice tonight," he said. "So now we have to check the data, but to understand if we fixed the problem we have to wait until tomorrow."