ABSTRACT The increasing threats to grassland ecosystems from land-use/land-cover change, disturbances, and invasive species underscore the importance of monitoring grassland plant diversity. While most remote sensing studies have mainly focused on quantifying α-diversity (diversity within communities), less attention has been given to remotely estimating β-diversity (diversity between communities) in naturally-assembled grasslands. In this study, we used remote sensing to map plant β-diversity in a naturally-assembled grassland managed using prescribed fire and grazing in a North American tallgrass prairie ecosystem within the US Southern Great Plains. We aimed to assess the impact of time since fire on the relationship between in situ plant β-diversity and remotely–sensed β-diversity, also known as spectral β-diversity. We collected in situ observations at 60 m × 60 m, 120 m × 120 m, 180 m × 180 m, and 240 m × 240 m plots, alongside airborne and spaceborne DESIS imaging spectroscopy data with spatial resolutions of 1 m and 30 m, respectively. To assess how management practices influenced β-diversity, we grouped our in situ observations into three categories based on time since fire, including recently-burned (burn age <1 year), transitional (burn age of 1–2 years), and unburned (burn age >2 years). Our findings showed that the association between in situ and spectral β-diversity was strongly influenced by time since fire. Excluding plots with high soil cover (≥50%) improved our remote estimation of β-diversity, despite soil exposure effects. Our study demonstrated that remote sensing of β-diversity in grassland ecosystems is influenced by spatial scale, time since fire, and soil exposure, all of which can be addressed with scale-appropriate imaging spectrometry. This study also highlighted the capability of spaceborne imaging spectrometers to estimate β-diversity of grassland ecosystems across large spatial domains.