Gatlin has run the three fastest sprints this year, leading the world 100m tables with 9.87sec from Beijing last month.
In Ostrava, the American will take on compatriot Mike Rodgers, veteran Kim Collins of St Kitts and Nevis, and South Africa's Simon Magakwe, who has a season's best of 9.98.
Gatlin is a replacement for the world's fastest man Bolt, who pulled out of Ostrava and the July 5 Diamond League meet in Paris in a bid to recoup training time after a foot injury.
Among female sprinters, four-time Olympic gold medallist and three-time world champion Felix will likewise replace out-of-form Shelly-Ann Fraser-Pryce of Jamaica.
Coming back from a hamstring injury that plagued her throughout the 2013 season, the American sprinter will face Jamaica's Aleen Bailey and France's Myriam Soumare over her preferred 200m.
Felix won the 200m sprint at the Diamond League event in Oslo last week in 22.73sec, after clocking a season best of 22.44sec two weeks earlier.
The Ostrava start list also features American sprinter LaShawn Merritt, who leads this year's 400m rankings with 43.97sec.
