Body of Abstract: Plant reproduction is sensitive to heat stress. Pollen tube growth can be accelerated or arrested by high temperatures, leading to unstable tubes, failed sperm cell delivery, and ultimately crop yield loss. Pollen growth dynamics have historically been observed on the scale of individual pollen grains, but there are only a few studies surveying pollen populations across genotypes and environmental conditions. We have developed a phenotyping system that quantifies tomato pollen characteristics on a large scale and under varied heat stress conditions. In this system, we combined high-throughput bright-field microscopy with automated object detection and tracking to quantify pollen phenotypes. We used this method to survey pollen from a diverse panel of 220 genome-sequenced tomato and close wild relative accessions. Pollen from these accessions showed a wide variety of responses to heat stress across measured phenotypes, suggesting genetic components influence heat stress responses that can be used to improve tomato productivity in challenging climates. This method can be readily adapted to pollen from different species, providing a way to rapidly characterize molecular mechanisms of pollen functions and identify thermotolerant alleles for enhanced reproduction in flowering plants.