Historic monuments and an upcoming marathon in the Czech capital Prague are under flood alert from the swollen Vltava river.
The Znojmo evacuations, prompted by rising water levels in the river Dyje, began Wednesday evening under the supervision of the emergency services.
Catastrophic floods hit the Znojmo region and Prague, with large parts of the country cut off or inundated, in August 2002, described as the worst in over a century.
Officials said the current flooding will likely turn out to be worse than the 2002 case.
The Znojmo evacuations, prompted by rising water levels in the river Dyje, began on Wednesday evening under the supervision of the emergency services.
Prague mayor Pavel Bem announced on Tuesday that the city was about to put into operation for the first time flood defence barriers set in place after 2002.
Metal flood barriers have been erected in the centre of Prague.
Several of Prague's most famous landmarks, including the National Theatre and the famous concert hall, the Rudolfinum, both sited near the river embankment, have also taken steps to prevent floodwater from seeping into their basements.
Both buildings suffered tens of millions of koruna damage in the last major floods.
The occupants of a handful of houses at Prague's low lying Zbraslav, around 10 kilometres from the centre of the capital have already been evacuated, and there is a chance that a further 100 might have to follow suit.
Around 20 roads near Pardubice, around 70 kilometres east of Prague, were closed by floodwaters on Wednesday.
The highest flood level alert, level three, had been declared at 45 sites across the country.
The move came amid a period of heavy rainfall and a swift rise in temperature following a very snowy winter.
An emergency meeting of the Czech government has been called for Thursday, with Prime Minister Jiri Paroubek cutting short a visit to Egypt to return to Prague.