AC Milan captain Montolivo was injured in the early stages of the friendly at Fulham’s Craven Cottage following a crunching tackle from Ireland defender Alex Pearce.
Skippering Italy on the night he needed several minutes of treatment and was seen mouthing “it’s broken” before eventually being carried off.
“Montolivo has what we suspected, a broken tibia,” Italy physician Enrico Castellacci said.
“I haven’t seen the X-ray yet to evaluate the extent of it but for a broken bone you can expect surgery.”
Montolivo played seven times in Italy’s World Cup qualifying campaign and had been expected to be a key member of the team in Brazil.
To make matters worse for Italian coach Cesare Prandelli, he also saw Fiorentina midfielder Alberto Aquilani, who had come on for Montolivo, forced off with an injury before half-time.
Prandelli, whose side kick off their World Cup campaign against England on June 14, has yet to prune his provisional 30-man squad down to a final group of 23 and he will now have to decide who will replace Montolivo.
In Rotterdam, Holland continued their build-up to the finals with a 1-0 win over fellow Brazil-bound Ghana, Manchester United striker Robin van Persie’s fifth minute goal proving decisive.
The Dutch, who open their World Cup campaign against titleholders Spain in a rematch of the 2010 final, have only one further prep lined up, against Wales on Wednesday in Amsterdam.
Van Persie’s goal came after a neat build-up between Wesley Sneijder and Arjen Robben.
The 2010 quarter-finalists Ghana were unable to produce any real chances against a stout Dutch defence but looked in good physical shape ahead of their opening World Cup game against the United States on June 14.
Dutch coach Louis van Gaal, who takes over at Manchester United after the World Cup, said: “We created quite a few chances even if the game wasn’t of great quality.
“I’m very happy with the way my new (5-3-2) system worked. For sure it’s not attractive as the old system of 4-3-3, you shouldn’t wait for a great show with 5-3-2.
“But up against Ghana, which is a great side, this system worked really well.
“There will be critics but I assume and I truly believe in this system and in my players chosen to carry it out.”
In Lisbon, Portugal, without injured star striker Cristiano Ronaldo (thigh), Pepe and Raul Meireles, played out a goalless draw with Greece.
The fixture was a rematch of the Euro 2004 final in the Portuguese capital which the hosts under then coach Luiz Felipe Scolari, now boss of Brazil, lost 1-0.
Portugal coach Paulo Bento said: “Tonight we couldn’t call on many players, not only Cristiano, and I reckon we put up a good display.”
Portugal open in Brazil against Germany, before the USA and then Ghana in Group G.
Croatia tuned up for their clash with hosts Brazil in the World Cup curtain-raiser with a 2-1 win over Mali, while in Oslo Fabio Capello’s Russia were held 1-1 by Norway.
Elsewhere, Algeria saw off Armenia 3-1 and Bosnia Herzegovina beat Ivory Coast 2-1 with Manchester City striker Edin Dzeko responsible for both of Bosnia’s goals.
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