Stefano Mauri, 32, was held along with former Genoa midfielder Omar Milanetto, 36, police said.
Juventus coach Antonio Conte, who just led the club to the Serie A title in his first season in charge, is among those being questioned by police.
Officers also visited Italy’s pre-Euro 2012 training camp to question left-back Domenico Criscito, 25.
Police are searching more than 30 homes, including those of players, trainers and administrators of clubs in Serie A, Serie B and lower divisions.
Five people were also arrested in Hungary on suspicion of being part of an illegal international betting ring.
Monday’s operation was part of a wider investigation which has already seen a number of arrests of current and former Italian players. In June last year, the Interior Ministry set up a special match-fixing task force in response to a number of high-profile cases.
Former Atalanta captain and Italy midfielder Cristiano Doni was banned for three-and-a-half years in August for his part in the “Calcioscommesse” scandal involving Serie B matches last season. He was also arrested in December 2011 over match-fixing and betting allegations.
Atalanta, promoted from Serie B, were deducted six points in the top flight this season as a result of the scandal.
Former Lazio and Italy striker Giuseppe Signori was banned for five years and 15 other players were banned for between one and five years for their parts.
Stefano Mauri, who joined the Rome club from Udinese in 2006 and has 11 caps for Italy, made 16 league appearances last season as Lazio finished fourth in Serie A.
Omar Milanetto signed for Serie B side Padova last summer after five years at Genoa and made 15 starts last season.
Domenico Criscito, a former Genoa and Juventus defender, is now at Zenit St Petersburg in Russia and has 19 caps for Italy.
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