Identification of genetic diversity in heat tolerance and associated markers is of great importance for improving heat tolerance in cool-season grass species. In this study, 98 accessions of perennial ryegrass (Lolium perenne L.) were subjected to heat stress (35/30 ℃, day/night) or optimal growth temperature (25/20 ℃) for 24 d in growth chambers. Overall heat tolerance of those accessions was ranked by principal component analysis (PCA) based on eight growth and physiological traits. Among these traits, chlorophyll (Chl) content had the highest correlation coefficient (0.864) with the PCA ranking of heat tolerance, indicating it was the most closely linked parameter to heat tolerance. And expressions of four Chl catabolic genes (CCGs) were negatively correlated with PCA ranking of heat. Furthermore, simple sequence repeat (SSR) markers were identified that significantly associated with Chl content and other heat tolerance-related traits. Together, the result highlighted the importance of Chl catabolism in heat tolerance of cool-season grasses. Chl content, heat-associated CCG genes and their associated SSR markers could be used as reliable trait or molecular markers in the breeding program of perennial ryegrass toward better heat tolerance.