Heat waves occur with more regularity and they adversely affect the yield of cool season crops including carrot (Daucus carota L.). Heat stress influences various biochemical and physiological processes including cell membrane permeability. Ion leakage and increase in cell permeability are indicators of cell membrane stability and have been used to evaluate the stress tolerance response in numerous crops and inform plant breeders for improving heat tolerance. No study has been published about the effects of heat stress on cell membrane stability and relative cell injury of carrot. Therefore, the present study was designed to estimate these stress indicators in response to heat stress at the early and late seedling developmental stages of 215 diverse accessions of wild and cultivated carrot germplasm. The article identifies the relationship between early and late stages of seedling tolerance across carrot genotypes and identifies heat-tolerant genotypes for further genetic analysis. Significant genetic variation among these stress indicators was identified with cell membrane stability and relative cell injury ranging from 6.3% to 97.3% and 2.8% to 76.6% at the early seedling stage, respectively; whereas cell membrane stability and relative cell injury ranged from 2.0% to 94.0% and 2.5% to 78.5%, respectively, at the late seedling stage under heat stress. Broad-sense heritability ranged from 0.64 to 0.91 for traits of interest under study, which indicates a relatively strong contribution of genetic factors in phenotypic variation among accessions. Heat tolerance varied widely among both wild and cultivated accessions, but the incidence of tolerance was higher in cultivated carrots than in wild carrots. The cultivated carrot accessions PI 326009 (Uzbekistan), PI 451754 (Netherlands), L2450 (USA), and PI 502654 (Pakistan) were identified as the most heat-tolerant accessions with highest cell membrane stability. This is the first evaluation of cell membrane stability and relative cell injury in response to heat stress during carrot development.