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The world silver medallist will face Olympic champion Pamela Jelimo of Kenya, who boasts the fastest time this season of one minute, 56.94 seconds (1:56.94) which she posted at the Doha Diamond League meeting two weeks ago.
Also in the field will be Fantu Magiso of Ethiopia, who finished second behind Jelimo in 1:57.90, the second fastest time of the season.
Semenya has shown slow progress this season but has not yet come up against world class athletes and a fast race can be expected on Friday.
She dipped below the Olympic qualifying standard of 1:59.90 for the first time this season at the final Yellow Pages meeting in Pretoria last month in a time of 1:59.58.
Meanwhile, Oscar Pistorius will continue his pursuit of reaching his first Olympic Games at the same meeting.
Pistorius ran his first Olympic qualifying time of 45.30 seconds in the 400m on March 17 at the Gauteng North Championships where he clocked 45.20.
The double-amputee sprinter needs to run the qualifying standard one more time to realise his dream.
Pistorius defended his Paralympic World Cup title in the 200m in Manchester on Tuesday.
A host of other South Africans, aiming to qualify for the Games, are included in Ostrava's preliminary startlist.
South African sprint sensation Simon Magakwe will go head-to-head with Jamaican world record holder Usain Bolt in the 100m.
Magakwe has already run the Olympic qualifying time of 10.18 six times in the 100m and has reached the 20.55 required in the 200m, twice.
The 27-year-old South African has been in superb form this season equalling the SA record of 10.06 earlier this year.
His time is the fourth fastest of all the athletes in the field, while Bolt posted a world leading time of 9.82 in Kingston, Jamaica earlier this month.
Other SA athletes also looking for their second qualifying times are, Hannes Dreyer, Thuso Mpuang and Tsholofelo Thipe.
Distance runner and SA 10km champion Elroy Gelant will be running in the 3 000m.
Mpuang needs to run a 200m qualifying time once more to qualify for the Games, and the same applies to Thipe in both the 200m and 400m.
Thipe will achieve her goal should she dip below 23.10 in the 200m and 51.55 in a 400m.
South African javelin record holder Sunette Viljoen will be looking to stamp her authority over the leading women in her specialist event.
Viljoen has already qualified for the Games at the SA Senior Championships in Port Elizabeth in April with a season's best heave of 61.15m.
She faces a tough field in world leading thrower Maria Abakumova of Russia, Slovenia's Martina Ratej and world record holder Barbora Špotkov of the Czech Republic, who have all thrown further than 65m this season.
Semenya, Viljoen, Willem Coertzen (decathlon), LJ van Zyl and Cornel Fredericks (400m hurdles) and Marc Mundell (50km race walk) have all already qualified for the Games in July.
