One of the major disfunctions that occurs in heat-stressed plants is enhanced protein damage and a consequent decline in cellular protein content. Creeping bentgrass (Agrositis stolonifera L.) is an economically important perennial grass species which is largely used on high value turf areas but experiences frequent damage due to heat stress. Several promising experimental lines of creeping bentgrass showed various tolerance levels to heat stress, with differential responses observed in physiological traits, total protein content and rates of protein degradation. The ubiquitin-proteasome system (UPS) plays a crucial role in the removal of damaged proteins, and there is a critical need to better understand the changes in protein accumulations and degradation via the UPS that occur during heat stress. Hence, we aimed to estimate change in global protein accumulations by performing gel-free proteomics as well as identify proteins that have been polyubiquitinated and targeted to the UPS pathway via polyubiquitin-omics in contrasting creeping bentgrass lines exposed to heat stress. It was found that heat-tolerant S11 729-10 was able to maintain less severe downregulation of proteins involved in the light reactions of photosynthesis, while enhancing the upregulation of antioxidant proteins, particularly during the later phase of stress. These contributed to its improved physiological performance including greater cell membrane integrity as well as healthier light harvesting components. Additionally, the faster turnover of key polyubiquitinated antioxidant proteins in S11 729-10 likely represents a critical mechanism for protecting against oxidative damage and enhancing tolerance under prolonged heat stress. This is the first time that the application of polyubiquitin-omics has been utilized in turfgrass. These findings provide deeper insights into protein metabolism underlying heat tolerance. Key stress-related traits or proteins identified in this study could be utilized to help develop new cultivars with enhanced tolerance to heat stress.