Bermudagrass [Cynodon dactylon (L.) Pers.] is a perennial and typical warm-season grass. It undergoes withering and severe damage under cold stress; thus, cold is considered as a key factor that restricts the widespread use in bermudagrass. Identification of association between molecular markers and cold tolerance-related traits would facilitate the efficient selection of cold tolerant bermudagrass cultivars. A total of 106 diverse bermudagrass accessions, including 4 commercial cultivars and 102 wild germplasms, were tested for cold tolerance and analyzed by 104 simple sequence repeat (SSR) markers. Cold significantly decreased transpiration rate, growth rate and turf quality. There were significant variations in these trait values among the accessions under cold conditions. Two subpopulations were detected in the panel of accessions based on the analysis of 1474 alleles with 104 SSR markers. Clustering analysis revealed that the genetic relationship was affected by the natural habitats. Thirty-four SSR markers were identified to be associated with two or three traits based on the corrected P values (P < 3.5 × 10^-4). These markers can be used for genetic improvement of cold tolerance of bermudagrass after further validation.