The fire regime is essential in creating a mosaic of plant structure and diversity in South American open savannas, especially favouring herbs. However, studies investigating diversity patterns in Neotropical savannas rarely focus on the herb–subshrub layer. This study investigated the variation of the herb–subshrub layer under contrasting fire regimes in the most conserved site within the Cerrado Domain, the Jalapão region, Brazil. We selected four sites of open savanna physiognomy with similar topographic, climatic and edaphic features: three burned every 2 years, while the fourth site has remained unburned for at least the last 10 years. We randomly distributed 15 plots of 4 m2 in each site and identified all herbs and subshrubs in each plot to estimate density, richness, alpha diversity and species composition. The unburned site had lower herb–subshrub density, richness and diversity than the frequently burned sites. Species composition varied between frequently burned and unburned sites, partially explained by the fire frequency across sites. Although other ecological factors may explain the patterns detected, we cannot rule out the importance of fire in structuring plant communities in the Jalapão region. As in other savannas, our study in the Cerrado Domain reinforces the essential role of the fire regimes in modifying and maintaining the diversity of herbaceous plants at the landscape scale.