Ferrari's Sebastian Vettel set the pace as Mercedes struggled in second practice at the Mexican Grand Prix.
Vettel was 0.614 seconds quicker than the fastest Mercedes of Valtteri Bottas in fourth, with Hamilton a further 0.349secs adrift in fifth.
Red Bull's Max Verstappen was second, 0.115secs behind Vettel, with Vettel's team-mate of Charles Leclerc third.
Mercedes' pace suggested Hamilton will struggle to tie up the title on Sunday - he needs 14 points more than Bottas.
To secure his sixth title, Hamilton at the very least needs to finish on the podium and hope Bottas' result goes his way.
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Friday practice is not always an accurate guide to true performance because it is impossible to know in what specification the teams are running their cars and fuel load and engine modes can make a significant difference to lap time.
But Mercedes came to Mexico City expecting to struggle because engine performance and straight-line speed, an area in which they have been left behind by Ferrari this season, is a major contributor to lap time.
The Mercedes was well over 10km/h slower than the Ferrari on the long pit straight and 0.5secs slower in the first sector, which features two straights and the first right-left-right chicane.
What about race pace?
But Mercedes looked much stronger on race pace, Hamilton quicker than Vettel on average over his race-simulation run on mediums tyres by about 0.15secs.
On soft tyres on a race stint, Verstappen was about 0.2secs quicker on average than Bottas, with Leclerc struggling well off the pace. Leclerc also had a harmless spin at the Esses late in the session.
Hamilton topped the first practice session in the morning by 0.119 seconds from Leclerc, but the Monegasque was using harder, slower tyres than the Briton.
Hamilton, whose regular race engineer Peter Bonnington is missing this race and the next one because of a medical procedure, sounded frustrated on the team radio when talking to his replacement Marcus Dudley.
Towards the end of the session, he sarcastically asked: "Are you going to give me any feedback." When told his pace was comparable to Bottas' in race trim and that the other drivers were on non-comparable runs, he said: "There is no grip."
"It's not been a great day, but this is how it usually is for us here," said Hamilton. "We have learnt a lot. It is just trying to understand the tyres. It is hard to pinpoint exactly how you get the tyres working. We are not massively off the Red Bulls but the Ferraris - they keep turning up their engine on the straights and the difference between us and them is massive on the straights."
It was a difficult session for Anglo-Thai rookie Alexander Albon in the second Red Bull. He crashed heavily early on and will start Saturday very much on the back foot on his first experience of the Autodromo Hermanos Rodriguez.
The Toro Rosso drivers Daniil Kvyat and Pierre Gasly were best of the rest in sixth and seventh, ahead of McLaren's Carlos Sainz, Renault's Nico Hulkenberg and McLaren's Lando Norris.
How Hamilton can win the title in Mexico
- Win plus fastest lap with Bottas lower than third
- Win with Bottas lower than fourth
- Second plus fastest lap with Bottas lower than seventh
- Second with Bottas lower than seventh
- Second with Bottas lower than eighth if Bottas sets fastest lap
- Third and fastest lap with Bottas lower than eighth
- Third with Bottas lower than ninth