Anwar has said that after March elections that handed the opposition alliance a third of parliamentary seats and five states, he is poised to oust the coalition with the help of defecting government lawmakers.
Analysts said the government would use the by-election to try to undermine Anwar's ability to appeal to all of Malaysia's racial groups - a first in the nation's political history.
"They will probably try to convince voters that Islam is under threat and that Malay rights are under threat," said Ibrahim Suffian from the Merdeka Centre research firm.
DAP leader Lim Kit Siang said the vote would be a referendum on the opposition's performance since March.
"It is not called the mother of by-elections for nothing. It is an indication of national politics and of our track record so far, it is a barometer of politics in Malaysia at large," he told AFP.
"If Anwar succeeds in pulling in a large number of Malay votes, it will boost the oppositions' standing and the road to forming the next government will be clearer."