Plant tolerance of freezing temperatures results from plastic and genetically-based responses to environmental cues that trigger biochemical processes related to seasonal dormancy. It is not advantageous from a fitness standpoint to maintain high cold tolerance throughout the year, so populations have adapted to their local environment by fine-tuning phenological traits, including cold tolerance. Quantifying variation for cold tolerance may be especially important for conservation or restoration efforts (e.g., assisted migration), as well as breeding programs for disease resistance (e.g., American chestnut, American elm) or crop improvement, in which progenitors may be adapted to different climates than the offspring. These and other research efforts in plant biology would benefit from high-throughput phenotyping of cold tolerance to parameterize climate suitability. Relative electrolyte leakage (REL) has been used for decades to provide a laboratory assessment of the degree of injury caused by exposure to cold temperatures. In the REL assay, replicates of tissue are exposed to increasingly cold temperatures in a test chamber and sequentially removed at specific minimum temperatures. When cells freeze and rupture, electrolytes are released and are detectable in solution with a conductivity meter. The data are commonly summarized on a relative basis as the temperature where 50% of the electrolytes have been released and cellular repair is unlikely. The laborious nature of handling thousands of samples necessary for the sequential temperature intervals, and the lack of commercially available multi-sensor equipment for measuring electrical conductivity has constrained widespread adoption of the REL approach. Recently, a new high throughput REL system for assessing plant cold tolerance was developed by the USDA Forest Service in Burlington Vermont . This has helped our group explore the potential of using REL for phenotyping variation in adaptation to seasonal environments, in addition to traditional growth and phenology measures. Using examples from forest trees (Ulmus americana) and overwintering crop species (Cicer arietinum and Pisum sativum), we describe the diagnostic features of the REL temperature response curve and estimation of its parameters for use in phenotyping. The temperature response curve is very similar to a dose response curve fitted with a logistic or sigmoid model. The curve is bounded by Ymin and Ymax and the temperature where any percentage of electrolyte leakage i.e., 10%, 20%, 50%, may be determined. Using cold tolerance assays, we demonstrate the system’s high-throughput application to phenotyping two staple leguminous crops, Cicer arietinum (chickpea) and Pisum sativum (common pea) for sensitivity to freezing and to predict critical injurious temperatures. We also report an example for the forest tree Ulmus americana, in which differences in cold tolerance were observed in selections sourced from across a latitudinal gradient when grown in a common garden.