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Anwar, a former deputy prime minister, was charged in August last year with allegedly sodomising a 24-year-old male former aide.
He faces up to 20 years in prison if convicted of sodomy, a crime in Muslim-majority Malaysia.
He has denied the charge which he says is part of a political conspiracy against him by the government aimed at undermining his three-party opposition alliance.
The alliance made massive gains in the March 2008 general elections against the ruling coalition which has ruled Malaysia uninterrupted since it won independence more than half a century ago.
Appealing to the high court to reject the case, Anwar’s lawyers argued that medical tests conducted on the aide after the alleged sodomy were inconclusive.
As a result, they had argued, trying him for sodomy would be tantamount to abusing the justice system.
However the judge rejected the argument, saying the prosecution may have other witnesses or evidence to prove its case.
He also rejected the argument that the case was a “malicious prosecution”.
Anwar’s lawyers have said they will take their case to Malaysia’s court of appeal.
The case is the second time that Anwar has been accused of sodomy.
He spent six years in prison between 1998 and 2004 after being convicted of corruption and of sodomising his former family driver.
Anwar insisted he had been framed and was freed when Malaysia’s top court overturned the sodomy conviction.